Available via license: CC BY-NC 4.0
Content may be subject to copyright.
Arnaud PAUL, et al.
1
TITLE
Bcd1p controls RNA loading of the core protein Nop58 during C/D box snoRNP
biogenesis
AUTHORS' FULL NAMES
Arnaud PAUL (1), Decebal TIOTIU (1), Benoît BRAGANTINI (2), Hélène MARTY (1), Bruno
CHARPENTIER (1,3), Séverine MASSENET (1) and Stéphane LABIALLE (1,4)
AFFILIATIONS
(1) Université de Lorraine, CNRS, IMoPA, F-54000 Nancy, France.
(2) Current address: Department of Biochemistry and Molecular Biology, Mayo Clinic,
200 First Street SW, Rochester, MN 55905, USA.
(3) bruno.charpentier@univ-lorraine.fr
(4) stephane.labialle@univ-lorraine.fr
RUNNING HEAD (50 characters)
Bcd1p controls early assembly of C/D snoRNPs
KEYWORDS (up to six)
C/D box snoRNA, small nucleolar ribonucleoprotein, small non-coding RNA, BCD1/Bcd1p
(ZNHIT6), NOP58/Nop58p, Saccharomyces cerevisiae
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
2
ABSTRACT (250 words)
Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins (C/D snoRNPs) is
guided by conserved trans-acting factors that act collectively to assemble the core proteins
SNU13/Snu13, NOP58/Nop58, NOP56/Nop56, FBL/Nop1 and box C/D small nucleolar
RNAs (C/D snoRNAs), in human and in yeast, respectively. This finely elaborated process
involves the sequential interplay of snoRNP-related proteins and RNA through the formation
of transient pre-RNP complexes. BCD1/Bcd1 protein is essential for yeast cell growth and for
the specific accumulation of box C/D snoRNAs. In this work, chromatin, RNA and protein
immunoprecipitation assays revealed the ordered loading of several snoRNP-related proteins
on immature and mature snoRNA species. Our results identify Bcd1p as an assembly factor
of C/D snoRNP biogenesis that is likely recruited co-transcriptionally and that directs the
loading of the core protein Nop58 on RNA.
INTRODUCTION
In eukaryotes, numerous trans-acting factors orchestrate the various steps of ribosome
biogenesis. The process starts in the nucleolus with precursor (pre)-ribosomal RNA (rRNA)
synthesis by RNA polymerase I that is further modified, folded and processed to generate
mature rRNAs. Hundreds of small nucleolar ribonucleoproteins (snoRNPs) are involved in
the process: C/D snoRNPs produce site-specific ribose 2'-O-methylation while H/ACA
snoRNPs catalyze pseudouridylation of rRNAs. Moreover, some snoRNPs (e.g. U3) are
involved in pre-rRNA endonucleolytic cleavage (for reviews, see Watkins and Bohnsack,
2012; Lafontaine, 2015). Each C/D snoRNP includes a specific box C/D non-coding RNA
acting as a guide for the specificity of the RNP enzyme and a common set of four core
proteins, SNU13(15.5kD)/Snu13, NOP58/Nop58, NOP56/Nop56 and the methyltransferase
Fibrillarin or FBL/Nop1, in human and in yeast, respectively. The biogenesis of C/D snoRNP
is a fascinatingly intricate process involving the temporal association of several factors in
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
3
large multiprotein pre-RNP complexes coupled with snoRNA maturation (for a review, see
Massenet et al., 2017). Concerning the U3 snoRNA in budding yeast, this includes the
removal of an intron (Myslinski et al., 1990). Numerous studies have been conducted
enabling several aspects of this highly conserved process to be elucidated. The RNA-binding
protein SNU13/Snu13 directly binds the kink-turn (K-turn) RNA structures formed by the
conserved C/D motifs ( Watkins et al., 2000; Marmier-Gourrier et al., 2003) and is required
for the subsequent binding of NOP56, NOP58 and then NOP1, as proposed based on
archaeal and vertebrate models (Omer et al., 2002; Watkins et al., 2002; McKeegan 2009;
Gagnon et al., 2012). Also, the heterodimerization of NOP56/Nop56p and NOP58/Nop58p
via their coiled-coil domains may help lock the snoRNP into a conformation compatible with
methyltransferase activity, as suggested by structural and functional analyses of archaeal
RNPs (Aittaleb et al., 2003; Rashid et al., 2003; Lin et al., 2011). Importantly, these
interactions do not occur autonomously in eukaryotic cells but are mediated by several
factors that intervene transiently during the biogenesis process. These so-called assembly
factors include the assembly platform NUFIP1/Rsa1p (McKeegan et al., 2007; Boulon et al.,
2008) and its stabilizing factor ZNHIT3/Hit1p (Rothé et al., 2014a; Bizarro et al., 2014;
Quinternet et al., 2016) that interact with SNU13/Snu13p to form a trimer that may represent
the initial module of C/D snoRNP assembly (Rothé et al., 2014b). NUFIP1/Rsa1p is assumed
to bridge the core proteins SNU13/Snu13 and NOP58/Nop58 by interacting directly with the
latter (Boulon et al., 2008; Rothé et al., 2014b). Another actor in the assembly is the R2TP
complex that cooperates with HSP90/Hsp90p in a cochaperone-chaperone system and
contains the proteins RUVBL1/Rvb1, RUVBL2/Rvb2, RPAP3/Tah1 and PIH1D1/Pih1 (Zhao
et al., 2005; Boulon et al., 2008; Zhao et al., 2008). The R2TP complex recruits Nop58p
independently from the snoRNA by direct interaction with Pih1p, and this interaction helps
stabilize Nop58p (Kakihara et al., 2014). An important step of the assembly process relies on
NUFIP/Rsa1p that competes with NOP58/Nop58p for interaction with PIH1D1/Pih1p, likely
leading to the dissociation of NOP58/Nop58p-PIH1D1/Pih1p complexes and to the loading of
NOP58/Nop58p to RNA-bound SNU13/Snu13p (Rothé et al., 2014b). Finally, the protein
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
4
BCD1/Bcd1 (also called ZNHIT6) was revealed to be a leading actor of the assembly
process when it was shown to be required for cell growth and for the specific accumulation of
box C/D snoRNAs in Saccharomyces cerevisiae (Peng et al., 2003; Hiley et al., 2005) and
for the maintenance of box C/D snoRNA levels in human cells (McKeegan et al., 2007). In
budding yeast, Bcd1p interacts nonspecifically with RNA and its N-terminus (amino acids 1 to
168 out of a total of 366 amino acids) is sufficient to maintain snoRNA expression and cell
viability (Bragantini et al., 2016). The N-terminus contains a zinc-finger domain (ZNF, amino
acids 1 to 45) that is highly homologous to the ZNF domain of Hit1p and that is
indispensable, but not sufficient, for protein stability and function (Bragantini et al., 2016).
Also, works mainly based on analyses of protein-protein interactions suggest that human
BCD1 is integrated in a network of interactions involving PIH1D1, NUFIP1, RUVBL1&2,
NOP58 and, albeit poorly, SNU13 (McKeegan et al., 2007; McKeegan et al., 2009; Bizarro et
al., 2014). By integrating these data in the complex series of snoRNP-related protein
interactions, some authors have proposed that these proteins form a protein-only pre-
snoRNP complex that scaffolds SNU13 and NOP58 core protein assembly (McKeegan et al.,
2007; Boulon et al., 2008; Bizarro et al., 2014).
In this work, we present data that fuel current models of eukaryotic C/D snoRNP biogenesis.
Using chromatin, RNA and protein immunoprecipitation, we document the ordered loading of
snoRNP-related proteins on RNA species. We also provide evidence that Bcd1p acts as a
master and early manager of this process by controlling the association of the core protein
Nop58 with a pre-snoRNP complex and its loading on C/D snoRNAs.
RESULTS
Bcd1p controls mature C/D snoRNA steady-state levels
In order to test the role of Bcd1p in vivo, we generated a GAL1::3HA-BCD1 strain by
inserting the GAL1 promoter followed by a 3xHA tag upstream of the BCD1 gene in the
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
5
genome of the S. cerevisiae BY4741 strain. The repression of Bcd1p expression was
completed in less than 30 minutes when the cells were shifted from a permissive galactose
medium (YPG) to a repressive glucose medium (YPD) (Fig. 1A). Using RT-qPCR, we
analyzed the steady-state levels of a selected series of C/D snoRNAs (Fig. 1B) in cells
maintained in permissive YPG medium or shifted to repressive YPD medium for 6 hours (Fig.
1C), or for 16 hours (Fig. 1D) i.e. when the cells completely ceased to grow. We used
random hexamers as primers for the reverse transcription reaction performed on total RNA
extracts. We then performed quantitative PCR using primers targeting snoRNA gene bodies
to measure the expression level of the total population of snoRNAs (i.e. mature plus
immature species) (Fig. 1B). We also used primers targeting sequences downstream of the
U3 and U14 gene bodies to specifically measure the expression level of immature, 3'-
extended snoRNA species. Concerning the U3 snoRNA, we also analyzed the RNA species
harboring the U3 intron using primers targeting the intronic sequence.
In agreement with previous data (Peng et al., 2003; Hiley et al., 2005), Bcd1p depletion
progressively induced a dramatic drop in the steady-state level of total snoRNA species; the
effect was likely specific to C/D snoRNAs as it was not observed with other RNA species
(Fig. 1C&D and Suppl. Fig. S1). Concerning immature snoRNAs, the 3'-extended pre-U3
species accumulated significantly at 6 hours, whereas the intron-bearing U3 and 3'-extended
pre-U14 species were not affected (Fig. 1C). Interestingly, the accumulation of 3'-extended
U3 species suggested that the kinetics of U3 snoRNA 3'-end processing was affected, as
has already been reported in yeast cells depleted in the core protein Nop58 (Kufel et al.,
2000) or in cells in which the RSA1 and HIT1 genes were deleted (Rothé et al., 2014a). At 16
hours, the reduction in the expression level of total but not immature RNA species suggested
that the presence of Bcd1p was important for the accumulation of mature C/D snoRNAs. To
validate this hypothesis, we analyzed snoRNA gene transcription levels by evaluating the
recruitment of the RNA polymerase II (polII) machinery at snoRNA genes by chromatin
immunoprecipitation (ChIP). As depicted in figure 1E, we detected no influence of Bcd1p
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
6
depletion on the interaction between Rpb1p, the largest subunit of the core polII machinery,
and C/D snoRNA gene loci. This result confirmed that Bcd1p did not control the synthesis of
C/D snoRNAs but rather influenced their stability.
Bcd1p associates with C/D snoRNA gene loci in a RNA-dependent manner
The core proteins Nop1 and Nop58 have already been detected in interaction with C/D
snoRNA gene loci (Morlando et al., 2004, Vincenti et al., 2007) suggesting that RNP
biogenesis may start early at the site of RNA transcription. In order to test whether Bcd1p is
involved in early co-transcriptional stages, we performed ChIP analyses using a strain
expressing TAP-tagged Bcd1p. We indeed detected interactions with C/D snoRNA gene loci
but not with the H/ACA snR32 locus (Fig. 2A). Moreover, RNase A/T1 treatments
significantly decreased the level of Bcd1p-chromatin interaction, an effect that was not
observed when we analyzed Rpb1p-chromatin interactions (Fig. 2A). Thus, this experiment
suggested that Bcd1p is associated with chromatin via snoRNAs during their transcription or
soon after. To confirm this possibility, we took advantage of the tight coupling between
snoRNP biogenesis and snoRNA 3'-end maturation machinery (Fatica et al., 2000; Morlando
et al., 2002). A previous work showed that snoRNAs that are efficiently polyadenylated
cannot engage normally in the biogenesis process to produce mature and functional
snoRNPs (Fatica et al., 2000). We performed ChIP experiments on BCD1-TAP cells
transformed with p416 expression plasmids (Mumberg et al., 1995) harboring the U14 gene
body flanked at its 3' end either by its natural DNA sequence (U14-wt) or by the
polyadenylation site of the CYC1 gene (U14-cyc1). We detected a robust Bcd1p-plasmid
interaction only when the bona fide 3' flanking region of the U14 gene was present (Fig. 2B).
When we conducted a similar experiment with the H/ACA snR36 gene body, the resulting
interaction signal was weak despite enhancement due to the presence of its natural 3'
sequence. This result suggested that Bcd1p is recruited to transcription sites only when
correct RNA maturation and RNP assembly are engaged. Finally, we tested whether Bcd1p
associates with snoRNA species by RNA IP (RIP). We indeed observed a significant
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
7
interaction between Bcd1p and C/D snoRNAs, but not between Bcd1p and H/ACA snoRNAs
(Fig. 2C). Also, Bcd1p interacted with the intron-bearing U3 and the 3'-extended U14
species, thereby corroborating the fact that the interaction occurred during the snoRNA
maturation process. Surprisingly, Bcd1p was not detected in association with 3'-extended U3
species, probably due to epitope masking or because the TAP sequence reduced the
stability of the complex. Indeed, it has been reported that splicing of the premature U3 occurs
before 3'-end trimming and 5'-cap trimethylation (Myslinski et al., 1990; Kufel et al., 2000;
Kufel et al., 2003; Rothé et al., 2017), and we checked that in our samples the U3 species
with mature 3' ends were spliced (Suppl. Fig. S2). In conclusion, Bcd1p was recruited in vivo
on C/D pre-snoRNAs in the close vicinity of their transcription site, likely during or soon after
transcription.
Bcd1p controls the interaction of several snoRNP-related proteins with C/D snoRNAs
To gain further insights into the snoRNP biogenesis process and Bcd1p function, we tested
the interaction between several snoRNP-related proteins and snoRNA species in the
presence or absence of Bcd1p expression. To this end, we inserted the GAL1 promoter in a
series of TAP-tagged strains followed by a 3xHA tag upstream of the BCD1 gene when it
was tolerated by the cells (in the case of NOP56-TAP, RSA1-TAP, RVB2-TAP and PIH1-
TAP), or we introduced FLAG-tagged expression vectors of recombinant genes in the
GAL1::3HA-BCD1 strain (for the expression of Snu13p, Nop1p and Nop58p).
When the cells were cultivated in permissive YPG medium (Fig. 3, white drawbars), we
observed that the interaction levels with total C/D snoRNA species were higher for core
proteins (Fig. 3A-D; RIP signals around 20% to 60%) than for assembly factors (Fig. 3E-G;
RIP signals around 0.5 to 10%), which is in agreement with the transient interaction status of
the latter. Interestingly, the Bcd1p interaction level was in the high range compared to the
other assembly factors tested (Fig. 2C; RIP signals between 8% and 15%). Also, the H/ACA
snoRNA snR32 exhibited a significant interaction (RIP signals between 1% and 10%) for all
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
8
proteins except for Nop56p (Fig. 3D) and Bcd1p (Fig. 2C). Interactions with H/ACA snoRNAs
have already been described for the assembly factors NUFIP1 (Boulon et al., 2008) and
PIH1D1 (Machado-Pinilla et al., 2012). Interactions between different classes of snoRNPs
have also been observed repeatedly (van Nues RW, et al. 2011; Kishore S. et al. 2013;
Schwartz S. et al. 2014; Gumienny R. et al. 2017; Dudnakova T. et al. 2018) and have been
suggested to reflect the presence of different snoRNPs on the same target rRNA, or the fact
that H/ACA and C/D box RNPs guide modifications to each other. Focusing on immature U3
species, we analyzed two features: the presence of the intron and the presence of a 3'
extension. Interestingly, Snu13p interacted efficiently with RNA harboring both features (Fig.
3A and Suppl. Table 1; RIP signals of respectively 9.8% and 5.3%), but this was clearly not
the case for the other core proteins that exhibited low RIP signal levels (Fig. 3B-D and Suppl.
Table 1; RIP signals ranging from 0.05% to 0.8%). This observation confirmed that Snu13p is
the first core protein to interact with RNA during biogenesis. Concerning assembly factors,
the interaction between the immature U3 species and Rsa1p, Pih1p or Rvb2p was not
detected or did not significantly differ from background levels (Fig. 3E-G and Suppl. Table 1).
Interestingly, Bcd1p generated an interaction with intron-bearing U3 species that was
stronger than the one generated by the core proteins Nop1, Nop56 and Nop58 (Fig. 2C and
Suppl. Table 1; RIP signal level of 9.5% compared to 0.8% or less). Thus, intron-bearing U3
species interacted mostly with Snu13p and Bcd1p, suggesting that these proteins are among
the first to be recruited to RNA during U3 biogenesis. The 3'-extended U14 species
corroborated the observations made with pre-U3 species: Snu13p generated the highest RIP
signal levels (around 50%), the other core proteins generated robust signals (15-30%) while
assembly factors generated very low interaction levels (0.14-2.6%).
The consequence of Bcd1p depletion was tested after a 6-hour shift of yeast cultures to
repressive YPD medium (Fig. 3, black drawbars). At that time, the steady-state level of the
C/D snoRNP-related proteins was not affected (Suppl. Fig. S3) while the level of the C/D
snoRNA species was moderately affected (Fig. 1C). Interactions with total snoRNA species
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
9
followed two different regimens: those that were mostly unchanged, i.e. involving Snu13p
and Nop56p (Fig. 3A&C), and the other ones that dropped significantly, with the exception of
a few signals associated with higher noise (i.e. snR52 in Fig. 3B&D and U14 in Fig. 3G). The
same tendency was observed for interactions involving the immature RNA species that
dropped or remained almost undetectable. Conversely, the 3'-extended U3 species, whose
expression level increased in absence of Bcd1p (Fig. 1C), interacted better with Snu13p (Fig.
3A). Finally, the association of the proteins with the H/ACA snoRNA snR32 was insensitive to
Bcd1p depletion, in agreement with the fact that these two molecules did not significantly
interact (Fig. 2C). Therefore, Bcd1p was necessary for the efficient interaction of a large
subset of snoRNP-related proteins with immature and total RNA species, which includes the
essential Nop1 and Nop58 core proteins. In the absence of Bcd1p expression, biogenesis
was critically altered since only the two core proteins Snu13 and Nop56 maintained their
association with C/D snoRNAs.
Bcd1p controls the interaction of Nop58p with an early pre-snoRNP complex
To clarify the function of Bcd1p, we first analyzed its snoRNP-related protein partners. As
already identified in human orthologs (McKeegan et al., 2007; McKeegan et al., 2009;
Bizarro et al., 2014), yeast Bcd1p associated with the assembly factors Rsa1p, Pih1p and
Rvb2p, and with the core proteins Snu13 and Nop58 (Suppl. Fig. S4). Interestingly, the
association of Bcd1p with the RNA-binding Snu13p and its direct partner Rsa1p was
independent of RNA (Fig. S4A&B). We then tested the consequence of Bcd1p depletion on
selected interactions between these snoRNP-related proteins. The assembly factor Rsa1p
contributes to the connection between Snu13p and the assembly machinery (Rothé et al.,
2014b) and our RIP analyses indicated that the Rsa1p-RNA interaction, but not the Snu13p-
RNA interaction, decreased in absence of Bcd1p expression (Fig. 3A&E). However, co-IP
experiments conducted in cells maintained in permissive YPG medium, or after a 6-hour shift
to repressive YPD medium, revealed that the Snu13p-Rsa1p interaction was not dependent
on the presence of Bcd1p (Fig. 4A). Previous works showed that the core protein Nop58 is
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
10
recruited to the pre-snoRNP complex via Pih1p and the R2TP complex (Gonzales et al.,
2005; Quinternet et al., 2015; Kakihara et al., 2014; Bizarro et al. 2014). While the
interactions between Nop58p-Pih1p and Pih1p-Rsa1p were not significantly affected by the
absence of Bcd1p (Fig. 4B&C), the interaction between Nop58p and Rsa1p dropped
significantly (Fig. 4D). Taken together, these data suggest that Bcd1p affects the association
of Nop58p with a protein-only pre-snoRNP complex involving Rsa1p. To confirm this
hypothesis, we first tested whether Nop58p was still recruited at snoRNA gene loci in the
absence of Bcd1p expression. ChIP assays showed that the interaction was indeed detected
in cells in which Bcd1p expression is repressed as well as in cells in which RSA1 or PIH1
genes used as controls were deleted (Fig. 4E). Thus, Bcd1p does not control the recruitment
of Nop58p at the sites of snoRNP biogenesis but rather during a subsequent step that is
important for loading Nop58p on RNA. Second, the data argue that human BCD1 may
incorporate a protein-only pre-snoRNP complex to perform its function (Bizarro et al. 2014).
To test whether yeast Bcd1p can function independently of RNA, we generated Bcd1p
protein truncations in order to dissociate the biogenesis function of Bcd1p from its capacity to
interact with RNA. A recent analysis showed that the N-terminal part of the Bcd1 protein
(amino acids 1-168) is sufficient to maintain the level of snoRNA expression and cell viability
(Bragantini et al., 2016). Based on sequence conservation and secondary structure
prediction using PsiPred (Jones, 1999), we defined two short N-terminal fragments (1-115
and 1-96) for further analyses. We transformed GAL1::3HA-BCD1 cells with a p416 vector
expressing a FLAG-tagged version of the fragments or the full length protein. When the cells
were shifted for 16 hours to repressive YPD medium, the expression of Bcd1p fragments
was sufficient to maintain cell growth (Fig. 5A) as well as the steady-state level of snoRNAs
(Fig. 5B). Conversely, RIP analysis showed that these fragments were unable to interact
efficiently with RNA in vivo (Fig. 5C). In conclusion, Bcd1p was required to efficiently
incorporate the core protein Nop58 into an early pre-snoRNP complex that included Rsa1p
and this function did not require a strong association with RNA in order to be fulfilled.
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
11
DISCUSSION
In recent years, a wealth of studies has revealed that eukaryotic C/D snoRNP biogenesis is a
finely elaborated process. But the role of some players, such as the protein BCD1/Bcd1, is
still poorly understood. After its discovery as an essential factor for cell growth and C/D
snoRNAs stability in yeast (Peng et al., 2003; Hiley et al., 2005) and in human cells
(McKeegan et al., 2007), the identification of the protein partners of BCD1 in human cells
(Boulon et al., 2008, McKeegan et al., 2007, Bizarro et al., 2014), suggested its integration in
an early, protein-only pre-snoRNP complex (Bizarro et al., 2014). Here, we made significant
progress in the understanding of yeast Bcd1p function.
Bcd1p is an assembly factor that likely acts co-transcriptionally
We collected evidence concerning the association of Bcd1p with immature snoRNA species
that starts in the close vicinity of their transcription sites (Fig. 1). Detection of Bcd1p at
chromatin was RNA-dependent (Fig. 2A) and likely reflected a function during the early steps
of snoRNP assembly, since we failed to detect an influence on C/D snoRNA gene
transcription (Fig. 1E). This strongly suggests that the initiation of C/D snoRNP biogenesis is
co-transcriptional, thereby corroborating previous clues (Morlando et al., 2004, Vincenti et al.,
2007; Rothé et al., 2014b; Grzechnik et al., 2018), and resembling what has been shown for
H/ACA snoRNP biogenesis (Fatica A. et al., 2002; Ballarino et al., 2005 ; Yang PK. et al.,
2005 ; Darzacq et al., 2006; Richard et al., 2006). Therefore, the data support the current
paradigm of RNP biogenesis in which the assembly machinery actively engages with its
substrates during - or right after - their synthesis, rather than RNA and core proteins being
released from their site of synthesis and then encountering assembly factors by diffusion
(Fischer et al., 2015).
Bcd1p favors the interaction of RNA-loaded Snu13p with Rsa1p and not Nop56p
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
12
Currently, it is well established that the RNA-binding protein Snu13 is required for the loading
of the other core proteins on C/D snoRNAs (Omer et al., 2002; Watkins et al., 2002;
McKeegan 2009; Gagnon et al., 2012) and that Rsa1p and its stabilizing factor Hit1p
associate with Snu13p to form a trimer that is assumed to represent the initial module of C/D
snoRNP assembly (Rothé et al., 2014b). One function of Rsa1p is to bridge the core proteins
Snu13 and Nop58 (Boulon et al., 2008; Rothé et al., 2014b), the latter initially being recruited
and stabilized by Pih1p and the R2TP complex (Kakihara et al., 2014). Here, we provide
additional information on the early steps of biogenesis. We observed that proteins Snu13 and
Bcd1 associated robustly with intron-bearing U3 snoRNAs (Fig. 2C&3A). Additionally, when
BCD1 gene expression was turned down, Snu13p was the only protein to continue to be
strongly recruited to intron-bearing U3 species and to accumulate on 3'-extended U3
species. We observed a similar, higher association of pre-U14 species with Snu13p than with
the other snoRNP-related proteins (Fig. 3A). Conversely, we observed a very poor
association of Rsa1p with immature U3 and U14 species (Fig. 3E), suggesting that the
integration of Rsa1p in pre-RNP occurs later than expected. Collectively, these observations
suggest that a pool of “free Snu13p” interacts early with immature C/D snoRNAs. This
challenges current biogenesis models mainly resulting from protein interaction studies and
suggests a different system dynamics in which Snu13p is recruited to RNA before the
assembly machinery. The association then has to be supplemented and/or replaced by the
recruitment of the assembly machinery to proceed to RNP biogenesis. Whether Snu13p-
RNA complexes are abortive or could recruit other factors is of interest as it questions
whether protein-protein assemblies can occur directly on RNA or have to be initially formed
in a RNA-free form. One drawback here is that the immunoprecipitation of Snu13p
necessitated the use of a vector expressing a FLAG-tagged recombinant protein, as tagging
the endogenous gene was not tolerated by yeast cells. Therefore, it could be argued that the
observation of a “free pool” of Snu13p bound to RNA is due to the ectopic overexpression of
recombinant Snu13p. However, the same phenomenon is likely observed, albeit indirectly,
with endogenous Snu13p. Indeed, upon Bcd1p depletion, Nop56p was still recruited to
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
13
snoRNAs (Fig. 3C), an observation made with cells expressing the endogenous Snu13p.
Interestingly, Nop56p recruitment on RNA likely occurred via Snu13p: previous yeast two-
hybrid analyses indeed showed that Snu13p interacts robustly with Nop56p, more strongly
than in its interaction with Nop58p and comparable to its interaction with Rsa1p (Boulon et
al., 2008). Taken together, these observations suggest that Snu13p alone recognizes the K-
turns of immature snoRNAs and is then joined by Nop56p through direct interaction in the
absence of Bcd1p expression. This calls for analyses to test whether Snu13p-Rsa1p and
Snu13p-Nop56p interactions are mutually exclusive. Interestingly, a genome-wide genetic
screen using genetic interaction mapping (GIM) revealed a positive fitness effect when the
invalidation of the RSA1 gene was combined with the downregulation of NOP56 gene
expression, and which was not observed with the NOP58 gene (C. Saveanu, personal
communication). This observation corroborates the idea that Rsa1p and Nop56p act
antagonistically. Therefore, it is tempting to suggest that a critical function of Bcd1p is to
channel Snu13p-RNA complexes towards a competent biogenesis path. Indeed, in the
absence of Bcd1p, the interaction of RNA with several snoRNP-related proteins including
Rsa1p dropped drastically and generated aberrant particles with snoRNAs associated
mostly, if not exclusively, with Snu13 and Nop56 core proteins.
Bcd1p regulates the interaction of Nop58p with a pre-snoRNP complex and snoRNAs
Our data strongly suggest that the key function of Bcd1p is intrinsically independent of its
capacity to associate with RNA (Fig. 5) even if a very transient level of association with RNA
may be sufficient for function. It is possible that the latter actually relates mostly to the
coordination of RNA synthesis and assembly that would facilitate RNP biogenesis but could
be unnecessary when cells are cultivated in laboratory conditions. Whether Bcd1p naturally
plays its role on RNA or not, this observation invited us to explore the impact of Bcd1p
depletion at the level of protein-protein interactions. First, the existence of a protein-only, pre-
snoRNP complex involving BCD1 has been proposed in works using human cells (Bizarro et
al. 2014) and we confirmed in yeast that Bcd1p associated with several snoRNP-related
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
14
proteins, including Snu13p and Rsa1p in an RNA-independent manner (Suppl. Fig. S4).
Second, it is believed that early in the assembly, Nop58p is dissociated from its R2TP
partner Pih1p through the competitive interaction of Rsa1p, in order to reach Snu13p (Rothé
et al., 2014b; Quinternet al., 2015). Here we present new observations concerning this
Pih1p-Nop58p-Rsa1p functional triad in the absence of Bcd1p expression: first, the proteins
were no longer able to reach RNA thus ending in the absence of Nop58p in the residual RNP
(Fig. 3D); second, we observed a significant drop in the level of Rsa1p-Nop58p interaction
(Fig. 4D). A tempting interpretation to reconcile these observations is that Bcd1p is required
to mediate the transfer of Nop58p from Pih1p towards a complex including Rsa1p and
maybe Snu13p. In its absence, such a complex would be either unstable or no longer
competent for RNA binding, e.g. due to Snu13p burying. In any case, the data presented
here are evidence that Bcd1p is required to deliver Nop58p on RNA, which in turn, is a
precondition for complete biogenesis and for the formation of mature and functional C/D
snoRNPs.
Getting closer to the detailed mechanism of action of Bcd1p will require further work. To fulfill
its function, Bcd1p may perform an unknown step in the assembly process, possibly
involving an as yet unidentified factor, a post-translational modification of a protein alone or
in a complex, or a conformational modification of a pre-RNP complex. In this line of thought,
the search for other assembly factors and the biochemical isolation of transient complexes is
a challenging but ongoing task that represents an invaluable and potent source of progress.
In conclusion, the data we present here underline the fact that Bcd1p behaves as a global
on-off switch for the loading the C/D box assembly machinery on snoRNAs. In this regard, it
would be of interest to dedicate future analyses to search for metabolic or stress pathways
that target and modulate Bcd1p function in order to coordinate C/D snoRNP biogenesis with
other cellular systems.
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
15
MATERIAL & METHODS
Yeast strains and plasmids
The yeast strains used are listed in Supplementary Table 2. Yeast cells were pre-cultured in
selective medium then grown in glucose-containing medium (YPD). The GAL1::3HA-BCD1
strain was maintained in galactose-containing medium and shifted to glucose-containing
medium for 6 or 16 hours to repress the GAL1 promoter when required. Cells were collected
at log phase (OD600nm = 0.8 to 1). Genetic modifications were generated by homologous
recombination. Derivatives of plasmid p416GPD-3XFLAG were built by inserting PCR-
amplified fragments between the BamHI and XhoI restriction sites. The plasmids used are
listed in Supplementary Table 3.
Yeast cell extract preparation and immunoprecipitation assays
Cell pellets were washed and resuspended in lysis buffer (150 mM KCl, 5 mM MgCl2, 0.05%
TRITON-X100, 20 mM Tris-HCl, pH 7.5) and lysed by bead beating. The lysates were
clarified twice by centrifugation at 10,000 g for 5 minutes, and were then added to IgG-
Sepharose beads (Sigma-Aldrich), anti-FLAG Agarose beads (Sigma-Aldrich) or Glutathione
Sepharose beads (GE Healthcare) used as control, and incubated for 2 h at 4◦C. Beads were
washed four times in lysis buffer. For RIP experiments, RNAs were extracted with phenol-
chloroform. For protein co-IP assays proteins were fractionated on 12.5% SDS-PAGE and
analyzed by Western blot according to standard procedures using anti-TAP (Peroxidase Anti-
Peroxidase soluble complex antibody, Sigma) anti-FLAG (Abcam), anti-HA (Roche) and anti-
Dps1p (kindly provided by C. Allmang-Cura and G. Eriani, IBMC, Strasbourg, France)
antibodies, and revealed by anti-rabbit IgG, Horseradish Peroxidase (HRP) conjugated
(Thermo Fisher Scientific) and ECL Prime Western blotting system (GE healthcare).
Quantification of Western blot signals was performed using Fusion Solo (Vilber Lourmat) and
FusionCapt Advanced software.
Chromatin immunoprecipitation (ChIP) assays
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
16
Cells were fixed with 1% formaldehyde for 10 minutes at room temperature, quenched with
0.125 M glycine and lysed in lysis buffer (Hepes-KOH 50 mM [pH 7.5], 500 mM NaCl, 1 mM
EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS). Chromatin was sonicated to an
average length of 200-500 bp. IgG-Sepharose beads or anti-FLAG Agarose beads were pre-
cleared in the presence of 20 g/ml BSA for 2 hours. Protein-DNA complexes were captured
on beads for 2 hours, washed twice with low salt buffer (Hepes-KOH 50 mM [pH 7.5], 50 mM
NaCl, 1 mM EDTA, 0.1% Triton X-100, 0.01% Na-deoxycholate, 0.05% SDS), followed by
washes with LiCl buffer (250 mM LiCl, 10 mM Tris-HCl [pH 8], 1 mM EDTA, 0.01% Igepal,
0.05% Na-deoxycholate) and IPP150 buffer (10 mM Tris-HCl [pH 8], 15 mM NaCl, 0.01%
Igepal). After digestion with proteinase K, reversal of the cross-links at 70°C overnight and
elution, DNA was extracted by Phenol-Chloroform.
Reverse transcription and quantitative PCR
Complementary DNA was generated using M-MLV Reverse Transcriptase (Invitrogen) and
random hexamers unless stated otherwise. Quantitative PCR using iTaq Universal
SYBRGreen supermix (Biorad) was performed on a StepOnePlus real-time PCR system
(Biorad) using a relative quantification and a standard curve method. Oligonucleotides are
listed in Supplementary Table 4.
Statistics and quantitative analyses
Data are reported as mean values and standard errors of the mean from at least three
biological replicates. The significance level using a paired two-tailed Student's t-test was set
to P values *P < 0.05, **P < 0.01 and ***P < 0.001.
SUPPLEMENTARY INFORMATION
Supplementary Table 1 (pdf). RIP signal levels of immatures snoRNA species and snoRNP-
related proteins.
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
17
Supplementary Table 2 (pdf). Yeast strains used in this study.
Supplementary Table 3 (pdf). Oligonucleotides used in this study.
Supplementary Table 4 (pdf). Plasmids used in this study.
ACKNOWLEDGMENTS
We are deeply grateful to Florence Schlotter for experimental advice and A. Visvikis for the
careful reading of the manuscript. We also thank C. Allmang-Cura and G. Eriani for the gift of
antibodies.
FUNDING
This work was supported by the Centre National de la Recherche Scientifique (CNRS), the
University of Lorraine (UL), the European Union and Region Lorraine (Fond Européen de
Développement Régional - FEDER) and the National Research Agency (ANR-11-BSV8-
01503; ANR-16-CE11-0032). A. Paul is a pre-doctoral fellow from the French “Ministère de
l’Enseignement Supérieur et de la Recherche ».
REFERENCES
Aittaleb M, Rashid R, Chen Q, Palmer JR, Daniels CJ, Li H. 2003. Structure and function of
archaeal box C/D sRNP core proteins. Nat Struct Biol 10: 256–263.
Ballarino M, Morlando M, Pagano F, Fatica A, Bozzoni I. 2005. The cotranscriptional
assembly of snoRNPs controls the biosynthesis of H/ACA snoRNAs in Saccharomyces
cerevisiae. Mol Cell Biol 25: 5396–5403.
Bizarro J, Charron C, Boulon S, Westman B, Pradet-Balade B, Vandermoere F, Chagot M-E,
Hallais M, Ahmad Y, Leonhardt H, et al. 2014. Proteomic and 3D structure analyses highlight
the C/D box snoRNP assembly mechanism and its control. J Cell Biol 207: 463–480.
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
18
Boulon S, Marmier-Gourrier N, Pradet-Balade B, Wurth L, Verheggen C, Jády BE, Rothé B,
Pescia C, Robert M-C, Kiss T, et al. 2008. The Hsp90 chaperone controls the biogenesis of
L7Ae RNPs through conserved machinery. J Cell Biol 180: 579–595.
Bragantini B, Tiotiu D, Rothé B, Saliou J-M, Marty H, Cianférani S, Charpentier B, Quinternet
M, Manival X. 2016. Functional and Structural Insights of the Zinc-Finger HIT protein family
members Involved in Box C/D snoRNP Biogenesis. J Mol Biol 428: 2488–2506.
Darzacq X, Kittur N, Roy S, Shav-Tal Y, Singer RH, Meier UT. 2006. Stepwise RNP
assembly at the site of H/ACA RNA transcription in human cells. J Cell Biol 173: 207–218.
Dudnakova T, Dunn-Davies H, Peters R, Tollervey D. 2018. Mapping targets for small
nucleolar RNAs in yeast. Wellcome Open Res 3: 120.
Fatica A, Morlando M, Bozzoni I. 2000. Yeast snoRNA accumulation relies on a cleavage-
dependent/polyadenylation-independent 3'-processing apparatus. EMBO J 19: 6218–6229.
Fatica A, Dlakić M, Tollervey D. 2002. Naf1 p is a box H/ACA snoRNP assembly factor. RNA
8: 1502–14.
Fischer U, Chari A. 2015. Assembly of RNPs: help needed. RNA 21: 613–614.
Gagnon KT, Biswas S, Zhang X, Brown BA, Wollenzien P, Mattos C, Maxwell ES. 2012.
Structurally conserved Nop56/58 N-terminal domain facilitates archaeal box C/D
ribonucleoprotein-guided methyltransferase activity. J Biol Chem 287: 19418–19428.
Gonzales FA, Zanchin NIT, Luz JS, Oliveira CC. 2005. Characterization of Saccharomyces
cerevisiae Nop17p, a novel Nop58p-interacting protein that is involved in Pre-rRNA
processing. J Mol Biol 346: 437–455.
Grzechnik P, Szczepaniak SA, Dhir S, Pastucha A, Parslow H, Matuszek Z, Mischo HE,
Kufel J, Proudfoot NJ. 2018. Nuclear fate of yeast snoRNA is determined by co-
transcriptional Rnt1 cleavage. Nat Commun 9: 1783.
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
19
Gumienny R, Jedlinski DJ, Schmidt A, Gypas F, Martin G, Vina-Vilaseca A, Zavolan M. 2017.
High-throughput identification of C/D box snoRNA targets with CLIP and RiboMeth-seq.
Nucleic Acids Res 45: 2341–2353.
Hiley SL, Babak T, Hughes TR. 2005. Global analysis of yeast RNA processing identifies
new targets of RNase III and uncovers a link between tRNA 5' end processing and tRNA
splicing. Nucleic Acids Res 33: 3048–3056.
Jones DT. 1999. Protein secondary structure prediction based on position-specific scoring
matrices. J Mol Biol 292: 195–202.
Kakihara Y, Makhnevych T, Zhao L, Tang W, Houry WA. 2014. Nutritional status modulates
box C/D snoRNP biogenesis by regulated subcellular relocalization of the R2TP complex.
Genome Biol 15: 404.
Kishore S, Gruber AR, Jedlinski DJ, Syed AP, Jorjani H, Zavolan M. 2013. Insights into
snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA
sequencing. Genome Biol 14: R45.
Kufel J, Allmang C, Chanfreau G, Petfalski E, Lafontaine DL, Tollervey D. 2000. Precursors
to the U3 small nucleolar RNA lack small nucleolar RNP proteins but are stabilized by La
binding. Mol Cell Biol 20: 5415–5424.
Kufel J, Allmang C, Verdone L, Beggs J, Tollervey D. 2003. A complex pathway for 3'
processing of the yeast U3 snoRNA. Nucleic Acids Res 31: 6788–6797.
Lafontaine DLJ. 2015. Noncoding RNAs in eukaryotic ribosome biogenesis and function. Nat
Struct Mol Biol 22: 11–19.
Lin J, Lai S, Jia R, Xu A, Zhang L, Lu J, Ye K. 2011. Structural basis for site-specific ribose
methylation by box C/D RNA protein complexes. Nature 469: 559–563.
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
20
Machado-Pinilla R, Liger D, Leulliot N, Meier UT. 2012. Mechanism of the AAA+ ATPases
pontin and reptin in the biogenesis of H/ACA RNPs. RNA 18: 1833–1845.
Marmier-Gourrier N, Cléry A, Senty-Ségault V, Charpentier B, Schlotter F, Leclerc F,
Fournier R, Branlant C. 2003. A structural, phylogenetic, and functional study of 15.5-
kD/Snu13 protein binding on U3 small nucleolar RNA. RNA 9: 821–838.
Massenet S, Bertrand E, Verheggen C. 2017. Assembly and trafficking of box C/D and
H/ACA snoRNPs. RNA Biol 14: 680–692.
McKeegan KS, Debieux CM, Boulon S, Bertrand E, Watkins NJ. 2007. A dynamic scaffold of
pre-snoRNP factors facilitates human box C/D snoRNP assembly. Mol Cell Biol 27: 6782–
6793.
McKeegan KS, Debieux CM, Watkins NJ. 2009. Evidence that the AAA+ proteins TIP48 and
TIP49 bridge interactions between 15.5K and the related NOP56 and NOP58 proteins during
box C/D snoRNP biogenesis. Mol Cell Biol 29: 4971–4981.
Morlando M, Ballarino M, Greco P, Caffarelli E, Dichtl B, Bozzoni I. 2004. Coupling between
snoRNP assembly and 3' processing controls box C/D snoRNA biosynthesis in yeast. EMBO
J 23: 2392–2401.
Morlando M, Greco P, Dichtl B, Fatica A, Keller W, Bozzoni I. 2002. Functional analysis of
yeast snoRNA and snRNA 3'-end formation mediated by uncoupling of cleavage and
polyadenylation. Mol Cell Biol 22: 1379–1389.
Mumberg D, Müller R, Funk M. 1995. Yeast vectors for the controlled expression of
heterologous proteins in different genetic backgrounds. Gene 156: 119–122.
Myslinski E, Ségault V, Branlant C. 1990. An intron in the genes for U3 small nucleolar RNAs
of the yeast Saccharomyces cerevisiae. Science 247: 1213–1216.
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
21
Omer AD, Ziesche S, Ebhardt H, Dennis PP. 2002. In vitro reconstitution and activity of a
C/D box methylation guide ribonucleoprotein complex. Proc Natl Acad Sci USA 99: 5289–
5294.
Peng WT, Robinson MD, Mnaimneh S, Krogan NJ, Cagney G, Morris Q, Davierwala AP,
Grigull J, Yang X, Zhang W, et al. 2003. A panoramic view of yeast noncoding RNA
processing. Cell 113: 919–933.
Quinternet M, Chagot M-E, Rothé B, Tiotiu D, Charpentier B, Manival X. 2016. Structural
features of the box C/D snoRNP pre-assembly process are conserved through species.
Structure 24: 1693–1706.
Quinternet M, Rothé B, Barbier M, Bobo C, Saliou J-M, Jacquemin C, Back R, Chagot M-E,
Cianférani S, Meyer P, et al. 2015. Structure/Function analysis of protein-proteiniInteractions
developed by the yeast Pih1 platform protein and its partners in box C/D snoRNP assembly.
J Mol Biol 427: 2816–2839.
Rashid R, Aittaleb M, Chen Q, Spiegel K, Demeler B, Li H. 2003. Functional requirement for
symmetric assembly of archaeal box C/D small ribonucleoprotein particles. J Mol Biol 333:
295–306.
Richard P, Kiss AM, Darzacq X, Kiss T. 2006. Cotranscriptional recognition of human intronic
box H/ACA snoRNAs occurs in a splicing-independent manner. Mol Cell Biol 26: 2540–2549.
Rothé B, Saliou J-M, Quinternet M, Back R, Tiotiu D, Jacquemin C, Loegler C, Schlotter F,
Peña V, Eckert K, et al. 2014a. Protein Hit1, a novel box C/D snoRNP assembly factor,
controls cellular concentration of the scaffolding protein Rsa1 by direct interaction. Nucleic
Acids Res 42: 10731–10747.
Rothé B, Back R, Quinternet M, Bizarro J, Robert M-C, Blaud M, Romier C, Manival X,
Charpentier B, Bertrand E, et al. 2014b. Characterization of the interaction between protein
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
22
Snu13p/15.5K and the Rsa1p/NUFIP factor and demonstration of its functional importance
for snoRNP assembly. Nucleic Acids Res 42: 2015–2036.
Rothé B, Manival X, Rolland N, Charron C, Senty-Ségault V, Branlant C, Charpentier B.
2017. Implication of the box C/D snoRNP assembly factor Rsa1p in U3 snoRNP assembly.
Nucleic Acids Res 45: 7455–7473.
Schwartz S, Bernstein DA, Mumbach MR, Jovanovic M, Herbst RH, León-Ricardo BX,
Engreitz JM, Guttman M, Satija R, Lander ES, et al. 2014. Transcriptome-wide mapping
reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA. Cell 159:
148-162.
van Nues RW, Granneman S, Kudla G, Sloan KE, Chicken M, Tollervey D, Watkins NJ.
2011. Box C/D snoRNP catalysed methylation is aided by additional pre-rRNA base-pairing.
EMBO J 30: 2420–30.
Vincenti S, De Chiara V, Bozzoni I, Presutti C. 2007. The position of yeast snoRNA-coding
regions within host introns is essential for their biosynthesis and for efficient splicing of the
host pre-mRNA. RNA 13: 138–150.
Watkins NJ, Bohnsack MT. 2012. The box C/D and H/ACA snoRNPs: key players in the
modification, processing and the dynamic folding of ribosomal RNA. Wiley Interdiscip Rev
RNA 3: 397–414.
Watkins NJ, Dickmanns A, Lührmann R. 2002. Conserved stem II of the box C/D motif is
essential for nucleolar localization and is required, along with the 15.5K protein, for the
hierarchical assembly of the box C/D snoRNP. Mol Cell Biol 22: 8342–8352.
Watkins NJ, Ségault V, Charpentier B, Nottrott S, Fabrizio P, Bachi A, Wilm M, Rosbash M,
Branlant C, Lührmann R. 2000. A common core RNP structure shared between the small
nucleoar box C/D RNPs and the spliceosomal U4 snRNP. Cell 103: 457–466.
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
23
Zhao R, Davey M, Hsu Y-C, Kaplanek P, Tong A, Parsons AB, Krogan N, Cagney G, Mai D,
Greenblatt J, et al. 2005. Navigating the chaperone network: an integrative map of physical
and genetic interactions mediated by the hsp90 chaperone. Cell 120: 715–727.
Zhao R, Kakihara Y, Gribun A, Huen J, Yang G, Khanna M, Costanzo M, Brost RL, Boone C,
Hughes TR, et al. 2008. Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain
R2TP complex activity that regulates snoRNA accumulation. J Cell Biol 180: 563–578.
Yang PK, Hoareau C, Froment C, Monsarrat B, Henry Y, Chanfreau G. 2005.
Cotranscriptional recruitment of the pseudouridylsynthetase Cbf5p and of the RNA binding
protein Naf1p during H/ACA snoRNP assembly. Mol Cell Biol 25:3295-304.
FIGURE LEGENDS
Figure 1.
Effects of Bcd1p depletion on RNA expression level and RNA polymerase II recruitment at
selected gene loci. (A) Yeast GAL::3HA-BCD1 cells were shifted from galactose YPG
medium to glucose YPD medium for the times indicated before total protein extraction and
Western blotting. The Bcd1p signal was monitored using an anti-HA antibody. The aspartyl-
tRNA synthetase Dps1p was used to control protein loading. (B) Scheme of the RT-qPCR
strategy that allowed quantification of total (mature plus immature) or immature snoRNA
expression levels. (C and D) Cultures of yeast GAL::3HA-BCD1 cells were maintained in
YPG medium or shifted to YPD medium for 6 hours (C) or 16 hours (D) before RT-qPCR
assays. Relative RNA expression level in YPG condition was set to 1. The ALG9 gene
coding mannosyltransferase was used as an endogenous control. (E) Cultures of yeast
RPB1-TAP x GAL::3HA-BCD1 cells were maintained in YPG medium or shifted to YPD
medium for 16 hours before chromatin immunoprecipitation (ChIP) assays. IP was performed
using IgG-Sepharose beads and the GAL::3HA-BCD1 strain was used as a control.
Figure 2.
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
24
Interaction of Bcd1p with selected snoRNA gene loci and transcripts. (A) ChIP assays were
performed on yeast BCD1-TAP cells (grey drawbars) or RPB1-TAP cells (white drawbars)
cultivated in YPD medium. The BY4741 strain was used as control and IP was performed
using an anti-TAP antibody. Before IP, cell extracts were treated or not with RNase A/T1 (+/-
). (B) BCD1-TAP cells were transformed with empty p416 vector or recombinant p416 vector
containing U14 (snR128) or snR36 gene bodies flanked in 3' by their natural genomic
sequence or by the polyadenylation sequence of the CYC1 gene. ChIP assays were
performed using IgG-Sepharose beads and the qPCR reaction targeted the p416 backbone.
(C) RNA immunoprecipitation (RIP) assays were performed on yeast BCD1-TAP cells
cultivated in YPD medium. The BY4741 strain was used as a control and IP was performed
using IgG-Sepharose beads. For immature snoRNA species, the levels of the RIP signals
and their significance in tagged versus control strain are given in Supplementary table 1.
Figure 3.
Interaction of C/D snoRNP-related proteins with selected snoRNAs. A GAL1::3HA-BCD1
strain transformed with p416 vectors expressing FLAG-tagged versions of Snu13p (A),
Nop1p (B) and Nop58p (D), or NOP56-TAP x GAL1::3HA-BCD1 (C), RSA1-TAP x
GAL1::3HA-BCD1 (E), RVB2-TAP x GAL1::3HA-BCD1 (F) and PIH1-TAP x GAL1::3HA-
BCD1 (G) cells were maintained in YPG medium (white drawbars) or shifted to YPD medium
for 6 hours (black drawbars). RIP assays were performed using anti-FLAG beads and IgG-
Sepharose beads, respectively and GSH beads as control. For each RNA species, statistical
analysis is presented only for the comparison of YPG versus YPD related signals. For
immature snoRNA species, the significance of the RIP signal in tagged versus control strain
is given in Supplementary Table 1.
Figure 4.
Effect of Bcd1p on the molecular associations of several snoRNP-related proteins. (A-D) Co-
IP analyses were performed in RSA1-TAP X GAL1::3HA-BCD1 (A,C,D) and PIH1-TAP X
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
25
GAL1::3HA-BCD1 (B) cells transformed with a p416 vector expressing a FLAG-tagged
version of Snu13p (A), Nop58p (B,D) and Pih1p (C) or an empty p416 vector as control.
Yeast cells were maintained in YPG medium or shifted to YPD medium for 6 hours and all
IPs were performed with an anti-FLAG antibody. When useful, a second IP was performed
by adjusting the cell extract volume to the change in protein expression level induced by the
shift in the medium (in B & D) in order to minimize this effect. The Dps1 protein was used to
control protein loading. The relative interactions in presence (white drawbars) or absence
(black drawbars) of Bcd1p were quantified in three independent experiments. (E) ChIP
assays were performed on GAL1::3HA-BCD1, GAL1::3HA-BCD1 x rsa1
or GAL1::3HA-
BCD1 x pih1
cells transformed with a p416 vector expressing a FLAG-tagged version of
Nop58p. The cells were maintained in YPG medium or shifted to YPD medium for 6 hours
and IP was performed with anti-FLAG beads or GSH beads as control.
Figure 5.
Effect of the expression of N-terminal fragments of Bcd1p on cell growth, on selected
snoRNA steady-state expression levels and association with selected snoRNAs. (A) The
GAL1::3HA-BCD1 strain was transformed with a p416 empty vector or p416 vectors
expressing FLAG-tagged versions of Bcd1p, either full length or N-terminal fragments. The
cells were maintained in YPG medium then shifted to YPD medium at an OD600nm of 0.05 for
15 hours and their growth was monitored. (B) The relative RNA content of the same cells
was analyzed when maintained in YPG medium after a 16-hour shift to YPD medium. The
ALG9 gene was used as an endogenous control. (C) RIP Bcd1p assays were performed on
the same cells cultivated in repressive YPD medium for 16 hours. The IP was performed with
anti-FLAG beads and signal levels obtained in cells transformed with an empty p416 vector
was set to 1.
SUPPLEMENTARY FIGURE LEGENDS
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
26
Figure S1.
The steady-state levels of the mRNA Act1, the snRNAs U1 and U6, the H/ACA snoRNA
snR36 and the rRNA 25S were not affected by Bcd1p depletion. Cultures of yeast GAL::3HA-
BCD1 cells were maintained in YPG medium (white drawbars) or shifted to YPD medium for
6 hours (black drawbars) or 16 hours (grey drawbars) before RT-qPCR assays. The ALG9
gene coding mannosyltransferase was used as an endogenous control and the relative RNA
expression level in YPG condition was set to 1.
Figure S2.
U3 intron splicing occurred prior to 3'-end nucleolytic maturation. (A) Cartoon presenting the
set of specific primers used for the RT and qPCR steps in order to discriminate the
maturation state at the 3' extremity and the splicing status in U3 RNA species. Reverse
transcription of total RNA extracts used random hexamers (N6), a specific primer annealing
in the 3'-extension of U3 (RT immature), or a stem-loop primer annealing in the 3' terminus of
the mature U3 species (RT mature). (B) The last primer exhibits a stem-loop structure in its 3'
region that prevents it from binding to U3 species whose 3' are not trimmed (Rothé B., et al.
2017). (C) An intronic U3 sequence was detected by qPCR on total (immature plus mature)
U3 templates, on immature U3 templates but not on mature U3 templates, showing that
intron splicing occurred before 3’-end trimming.
Figure S3.
Bcd1p depletion had no effect on the expression level of snoRNP-related protein. Yeast
BY4741 or GAL1::3HA-BCD1 cells were transformed with a p416 vector expressing a FLAG-
tagged version of Snu13p, Nop1p or Nop58p (A-C). For the other tests, NOP56-TAP or
NOP56-TAP X GAL1::3HA-BCD1 (D), RSA1-TAP or RSA1-TAP X GAL1::3HA-BCD1 (E),
RVB2-TAP or RVB2-TAP X GAL1::3HA-BCD1 (F) and PIH1-TAP or PIH1-TAP X
GAL1::3HA-BCD1 (G) cells were used. For each experiment, the cells were maintained in
YPG medium or shifted to YPD medium for 6 hours before total protein extraction and
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
Arnaud PAUL, et al.
27
Western blotting. Protein signals were monitored using anti-FLAG, anti-TAP and anti-HA
antibodies. The Dps1 protein was used to control protein loading.
Figure S4.
Association of Bcd1p with selected snoRNP-related proteins. Co-immunoprecipitations were
performed on GAL1::3HA-BCD1 cells transformed with an empty p416 vector or p416
vectors expressing FLAG-tagged versions of Snu13p (A), Rsa1p (B), Nop58p (C) and Pih1p
(D). For the detection of Rsa1p (B) and Rvb2p (E) interactions, due to the intense
background signal obtained with these proteins, we used a RSA1-TAP or a RVB2-TAP
strain, respectively, transformed with an empty p416 vector or a p416 vector expressing a
FLAG-tagged version of Bcd1p. The associations of Bcd1p with the snoRNA-binding protein
Snu13p and its cofactor Rsa1p were also tested for their dependency on RNA (A & B), and
the effect of RNases A/T1 treatment on interaction levels was quantified (N=3). IPs were
performed using anti-FLAG beads and the Dps1 protein was used to control protein loading.
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
published online January 30, 2019RNA
Arnaud Paul, Decebal Tiotiu, Benoît Bragantini, et al.
box snoRNP biogenesis
Bcd1p controls RNA loading of the core protein Nop58 during C/D
Material
Supplemental http://rnajournal.cshlp.org/content/suppl/2019/01/30/rna.067967.118.DC1
P<P
Published online January 30, 2019 in advance of the print journal.
Manuscript
Accepted
manuscript is likely to differ from the final, published version.
Peer-reviewed and accepted for publication but not copyedited or typeset; accepted
License
Commons
Creative
.http://creativecommons.org/licenses/by-nc/4.0/
(Attribution-NonCommercial 4.0 International), as described at
After 12 months, it is available under a Creative Commons License ).http://rnajournal.cshlp.org/site/misc/terms.xhtmlthe full-issue publication date (see
This article is distributed exclusively by the RNA Society for the first 12 months after
Service
Email Alerting
click here.the top right corner of the article or
Receive free email alerts when new articles cite this article - sign up in the box at
http://rnajournal.cshlp.org/subscriptions
go to: RNATo subscribe to
Published by Cold Spring Harbor Laboratory Press for the RNA Society
Cold Spring Harbor Laboratory Press on January 31, 2019 - Published by rnajournal.cshlp.orgDownloaded from
