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Computational and Structural Biotechnology Journal
journal homepage: www.elsevier.com/locate/csbj
Research article
Phosphorylation of pICln by the autophagy activating kinase ULK1
regulates snRNP biogenesis and splice activity of the cell
Lea Marie Esser
a
, Katharina Schmitz
a
, Frank Hillebrand
b
, Steffen Erkelenz
b
, Heiner Schaal
b
,
Björn Stork
a
, Matthias Grimmler
c,1
, Sebastian Wesselborg
a
, Christoph Peter
a,⁎
a
Institute of Molecular Medicine I, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
b
Institute of Virology, University Hospital Düsseldorf, Düsseldorf, Germany
c
HOCHSCHULEN FRESENIUS gem. Trägergesellschaft mbH, University of Applied Sciences, Limburger Straße 2, 65510 Idstein, Germany
article info
Article history:
Received 15 September 2022
Received in revised form 10 March 2023
Accepted 11 March 2023
Available online 16 March 2023
Keywords:
Autophagy
PRMT5
pICln
ULK1
UsnRNP
spliceosomal activity
abstract
The spliceosome, responsible for all mature protein-coding transcripts of eukaryotic intron-containing
genes, consists of small uridine-rich nuclear ribonucleoproteins (UsnRNPs). The assembly of UsnRNPs de-
pends, on one hand, on the arginine methylation of Sm proteins catalyzed by the PRMT5 complex. On the
other hand, it depends on the phosphorylation of the PRMT5 subunit pICln by the Uncoordinated Like
Kinase 1 (ULK1). In consequence, phosphorylation of pICln affects the stability of the UsnRNP assembly
intermediate, the so-called 6 S complex. The detailed mechanisms of phosphorylation-dependent integrity
and subsequent UsnRNP assembly of the 6 S complex in vivo have not yet been analyzed.
By using a phospho-specific antibody against ULK1-dependent phosphorylation sites of pICln, we vi-
sualize the intracellular distribution of phosphorylated pICln. Furthermore, we detect the colocaliphosphor-
pICln1 with phospho-pICln by size-exclusion chromatography and immunofluorescence techniques. We
also show that phosphorylated pICln is predominantly present in the 6 S complex. The addition of ULK1 to
in vitro produced 6 S complex, as well as the reconstitution of ULK1 in ULK1-deficient cells, increases the
efficiency of snRNP biogenesis. Accordingly, inhibition of ULK1 and the associated decreased pICln phos-
phorylation lead to accumulation of the 6 S complex and reduction in the spliceosomal activity of the cell.
© 2023 Published by Elsevier B.V. on behalf of Research Network of Computational and Structural
Biotechnology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
1. Introduction
Pre-mRNA splicing in eukaryotes is catalyzed by the spliceosome,
a multimegadalton complex comprised of five small uridine-rich
nuclear ribonucleoproteins (UsnRNPs) [1,2]. Each UsnRNPs consists
of a specific UsnRNA (U1, U2, U4, U5, or U6) and, except for the U6
snRNP, a common set of seven Sm proteins (B, D1, D2, D3, E, F, and
G), which form a ring-shaped structure around the UsnRNA and thus
form the functional snRNP [1,3]. The assembly of Sm proteins and
their respective snRNAs is mainly arranged by the cooperated action
of two multiprotein complexes: the PRMT5 (Protein arginine N-
methyltransferase 5), also called methylosome, and the SMN (Sur-
vival of motor neuron) complex [4–9]. The PRMT5 complex, con-
sisting of the Protein Arginine Methyltransferase 5 (PRMT5), the
WD-repeat protein WD45, and pICln, catalyzes the arginine me-
thylation of the Sm proteins B, D1, and D3 and subsequently trans-
fers them onto the SMN complex [4,8–10]. The SMN multiprotein
complex consists of the survival of motor neuron (SMN) protein, its
binding partners known as Gemins 2–8, and the UNR interacting
protein (UNRIP) [5,11–15]. The SMN protein and Gemin2 form the
functional core of the SMN complex that is responsible for binding
the Sm proteins [15–17]. The interaction of the PRMT5 and the SMN
multiprotein complexes in the assembly of snRNPs is a tightly
regulated process in which pICln attributes a key role: on the one
hand it recruits the Sm proteins as substrates for PRMT5 and on the
other hand it also functions as an assembly chaperone by forming a
stable ring-shaped RNA-free intermediate with the Sm proteins D1,
D2, E, F, and G. This structure is called the 6 S complex [18,19]. To
form functional snRNP, pICln must be released from this inter-
mediate 6 S ring structure and needs to be replaced by the two
missing Sm proteins B and D3. It has been speculated that this key
substitutional step may be regulated by phosphorylation processes
Computational and Structural Biotechnology Journal 21 (2023) 2100–2109
https://doi.org/10.1016/j.csbj.2023.03.015
2001-0370/© 2023 Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
]]]]
]]]]]]
⁎
Corresponding author.
E-mail address: christoph.peter@uni-duesseldorf.de (C. Peter).
1
Present address: DiaSys Diagnostic Systems GmbH, Holzheim, Germany
[18]. In our recent work, we have shown that the autophagy acti-
vating Ser/Thr Unc-51-like kinase (ULK1) catalyzes the phosphor-
ylation of specific serine residues of pICln in its C-terminus and
mediates the release of Sm Proteins onto the SMN complex upon
phosphorylation [20].
In our work presented here, we focus on the characterization of
the phospho-status and cellular distribution of endogenous pICln
and its influence on the efficiency of new synthesis of UsnRNPs, by
using a phospho-pICln-specific antibody. We also address the sub-
sequent ULK1-mediated /phospho-pICln-specific spliceosomal ac-
tivity in vivo.
2. Experimental procedures
2.1. Antibodies
The following primary antibodies were used for immunoblotting
and immunofluorescence: α-pICln C5 (sc-130668, Santa Cruz,
mouse), Antibody recognizing phosphorylated pICln generated by
Eurogentec (rabbit), Antibody recognizing pICln has been described
previously (21, rabbit), α-SMN (05–1532, Merck, mouse), α-SmE
(NBP243792, Novus Biologicals, rabbit), α-SmF (SAB2102258, Sigma
Aldrich, rabbit), α-SmG (PA5–49365, Invitrogen, rabbit), α-SmD1
(AV40693, Sigma Aldrich, rabbit), α-SmD2 (SAB2102257, Sigma
Aldrich, rabbit), α-2,2,7-Trimethylguanosin (MABE302, Merck,
mouse), α-ULK1 (8054; CST, rabbit), α-ULK1 (HPA063990; Prestige
Antibodies Sigma Aldrich, rabbit), α-ULK1 F4 (sc-390904; Santa Cruz,
mouse). The detection of proteins was carried out with the following
fluorescent secondary antibodies: IRDye 680LT goat α-rabbit, IRDye
680LT goat α-mouse, IRDye 800CW donkey α-rabbit, IRDye 800CW
donkey α-mouse. For the detection of proteins in vivo via IF the
following secondary antibodies were used: Goat anti-Mouse IgG (H
+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A11001,
Invitrogen), Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed
Secondary Antibody, Alexa Fluor 647 (A31573, Invitrogen).
2.2. Plasmids and proteins
ULK1/2 inhibitor MRT67307 was obtained through the MRC PPU
Reagents and Services facility (MRC PPU, College of Life Sciences,
University of Dundee, Scotland, mrcppureagents.dundee.ac.uk).
For all in vitro assays, the used plasmids were described pre-
viously [20].
To analyze the influence of the ULK inhibition on the cellular
splicing process, transient transfection with the following plasmids
were used: SV SRSF2 (2X) SD1 ∆vpuenv-eGFP, SV SRSF2 (2X) − 1G3U
∆vpuenv-eGFP (for cloning strategy see [21,22]); pUCB∆U1, pUCU1
6 A (for cloning strategy see [21]). pXGH5 expressing the human
growth hormone 1 (hGH1) under control of the mouse me-
tallothionein-1 promoter was co-transfected for normalization and
to monitor transfection efficiencies.
2.3. Cell lines and cell culture
All cells were cultivated at 37 °C, 5% CO
2
in a humidified area in
Dulbecco´s Modified Eagle Medium (4,5 g/L D-glucose, 41965–039,
Gibco) supplemented with 10% (v/v) Fetal Bovine Serum (FCS)
(F0804, Sigma Aldrich), 100 U/ml Penicillin and 100 µg/ml
Streptomycin (15140–122, Sigma Aldrich). HEK293T cells for im-
munofluorescence were seeded one day before the treatment with
1 × 10
5
per well. For knockdown of ULK1 for immunofluorescence
analysis, HEK293T cells were transfected in 24-well plates using
DharmaFECT1 (T-2001–02, GE Dharmacon) with 50 nM ULK1 siRNA
(L-005049–00–0010, SMARTpool, ON-TARGETplus, GE Dharmacon)
or 50 nM of the On-TARGET plus Non-targeting Control Pool (D-
001810–10–20, GE Dharmacon) for 72 h. For inhibition of ULK1 in
immunofluorescence analysis, cells were pre-incubated for 5 h with
30 µM ULK1 inhibitor MRT67307. For splicing assays, HEK293T cells
were seeded in six-well plates with 2.5 × 10
5
cells per well. Before
transfection, cells were pre-incubated for three hours with 30 µM of
the ULK inhibitor MRT67307. Transient transfection was carried out
by using the TransIT-LT1 reagent (Mirus Bio LLC) following the
manufacturer’s instructions and described previously [23]. Cells
were harvested for total RNA isolation 20 h after ULK inhibitor ad-
dition. MEF ULK1/DKO cells were reconstituted as described be-
fore [20].
2.4. Protein expression and purification
Recombinant proteins were overexpressed in BL21 competent E.
coli at RT for 4 h after induction with 1 mM IPTG. For cell lysis, the
lysis buffer containing 300 mM NaCl, 50 mM Tris/HCl pH 7.5, 5 mM
EDTA, 5 mM EGTA, 0.01% (v/v) Igepal, protease inhibitor cocktail
(4693132001, Roche), 50 mg/ml Lysozyme (12650–88–3, Serva) was
used as well as sonication. After centrifugation at 15,000 rpm for 1 h,
the lysate was incubated with Glutathione-Sepharose 4B
(17–0756–01, Cytivia) for 1.5 h at 4 °C and subsequently washed 3
times with lysis buffer before use in in vitro assays.
2.5. Cytoplasm extraction (S100) and size exclusion chromatography
HEK293T cells were incubated with Roeder A buffer [24] in three
times cell volume for 10 min at room temperature, dounced 10 times
with a tight douncer, and adjusted to a NaCl concentration of
150 mM. After centrifugation at 13,000 rpm for 30 min, the super-
natants (S100 extracts) were filtrated with Millex-HA, 0.45 µm filter
unit (Merck Millipore) and either used for immunopurification or
applied to a Superdex 200 HiLoad 16/600 or Superdex 200 increase
10/300 GL column (GE Healthcare). For size exclusion chromato-
graphy 1 ml of the sample was fractionated in running buffer
(150 mM NaCl, 50 mM Tris/HCl pH 7.5) and 0.5 ml fractions were
collected and analyzed by immunoblotting. The columns were cali-
brated with thyroglobulin (669 kDa), ferritin (440 kDa), aldolase
(158 kDa), and RNase (14 kDa) (GE Healthcare).
2.6. Immunoblotting and immunopurification
The protein concentration of S100 cytoplasm extract was mea-
sured by the Bradford method. The samples were separated by Tris/
Tricine or Tris/Glycine SDS gel electrophoresis [25] and transferred
to PVDF membranes (Immobilon-FL, Merck Millipore). For im-
munoblot analysis, indicated antibodies and signals were detected
with an Odyssey LI-COR Imaging System. For endogenous im-
munopurification, Protein-G-Sepharose (GE17–0618–01, GE Health-
care) was coated with 1 µg of the specific antibody for 1 h rotating.
Afterward, pre-cleared lysates were bound to the Sepharose for 3 h
at 4 °C. Purified proteins were washed 3 times with washing buffer
(50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% Igepal, 2.5 mM
MgCl
2
, 0.8 U/µl RNase Inhibitor (N2611, Promega) and protease in-
hibitor cocktail (4693132001, Roche). The elution was obtained in
sample buffer (375 mM Tris pH 7.5; 25.8% (w/v) glycerol; 12.3% (w/v)
SDS; 0.06% (w/v) Bromophenol blue; 6% (v/v) β-mercaptoethanol;
pH 6.8) and analyzed by immunoblotting.
2.7. Immunofluorescence microscopy
For Immunofluorescence 1 × 10
5
HEK293T cells per well were
seeded on coverslips in 24-well plates in a humidified area at 37 °C,
5% CO
2
in Dulbecco´s Modified Eagle Medium (4.5 g/L D-glucose,
41965–039, Gibco) supplemented with 10% (v/v) FCS (F0804, Sigma
Aldrich), 100 U/ml Penicillin and 100 µg/ml Streptomycin
(15140–122, Sigma Aldrich) one day before staining. On the next day,
L.M. Esser, K. Schmitz, F. Hillebrand et al. Computational and Structural Biotechnology Journal 21 (2023) 2100–2109
2101
cells were fixed with 4% paraformaldehyde for 10 min and per-
meabilized with 0,2% Triton X-100, after these steps cells were wa-
shed three times with Dulbecco´s phosphate-buffered saline (DPBS,
14190–094, Gibco). Blocking of the cells was carried out with 5% BSA
for 1 h at RT. For detection of pICln total, (both antibodies 1:500),
phospho-specific pICln (1:500), and ULK1 total (both antibodies
1:500) cells were incubated for 2 h. For colocalization experiments
with pICln-phospho, which is an antibody with rabbit host, and
ULK1 F4 (sc-390904) antibody, which is a mouse host, were co-
stained. For comparison as a control pICln total antibody from also
rabbit host was used. To control whether ULK1 siRNA worked and if
the inhibition or knockdown of ULK1 total has an effect on pICln
total localization the following antibodies were used: ULK1 Prestige
Antibody Sigma (HPA063990) rabbit host and pICln C5 antibody
Santa Cruz mouse host (sc-130668). For immunofluorescence
quantification pICln phospho/total the pICln C5 antibody was co-
stained with the phospho-specific one. The secondary Alexa Fluor
488 and Alexa Fluor 647 antibodies were incubated for 1 h and the
DNA was stained with DAPI. Microscopy was carried out with an
Axio Observer microscope from Zeiss with an ApoTome.2 and a 40 x
oil immersion objective.
2.8. Immunofluorescence quantification with Fiji
For the quantification of the intensity ratio between phospho-
specific pICln and pICln total, a macro was written in Fiji software
which measured the intensity of pICln total and pICln phospho in the
area of each cell. Therefore, the macro recognized pICln total staining
which is distributed all over the cell as the area of the cell, and the
DAPI staining as the area of the nucleus. The total area of the cell was
divided by the total DAPI count per picture which in consequence is
the relative area of the cell per picture. Subsequently, the measured
intensity for pICln total and phospho-specific pICln each was divided
by the relative area of the cell which results in the relative fluores-
cence intensity of those two stainings per cell. For calculation, the
value of the pICln phospho signal per cell was normalized on the
value of the pICln total. Afterward, the values for MRT67307 treat-
ment were normalized on HEK wt value, and the values for ULK1
siRNA were normalized on Non-targeting control. For every condi-
tion at least 300 cells were analyzed. The diagram and the calcula-
tion of the standard deviation as well as the statistics were made in
Origin Software. To test the significance of the values the data were
analyzed using a Mann-Whitney U test where the p value was below
0.005, meaning that the samples were significantly different from
each other.
2.9. In vitro translation and interaction assay
The proteins were [
35
S] methionine-labelled (Hartmann Analytic)
by using the TNT Quick Coupled Transcription/Translation System
(Promega). For interaction assay in vitro translated proteins were
incubated with GST fusion proteins purified with Glutathione-
Sepharose 4B (17–0756–01, Cytivia) rotating for 1.5 h at 4 °C in in-
teraction buffer (300 mM NaCl, 50 mM Tris/HCl pH 7.5, 1 mM EGTA,
1 mM EDTA, 1 mM DTT, and 0.01% (v/v) Igepal). The GST fusion
proteins were washed 2 times with interaction buffer and eluted by
adding sample buffer, following SDS-PAGE, coomassie staining, and
autoradiography.
2.10. In vivo labelling
For in vivo labelling cells were seeded in six-well plates in DMEM
(4.5 g/L D-glucose, 41965–039, Gibco) supplemented with 10% FCS
(F0804, Sigma Aldrich) with a density of 1 × 10
6
cells per well. The
cells were starved in media without methionine and cysteine
(D0422, Sigma Aldrich) for 30 min. For the labelling cells were
incubated with 100 µl media containing 10% dialyzed FCS, 20 mM
Hepes pH 7,4, and 25 µCi/ml [
35
S] methionine-label for 3.5 h at 37 °C.
Afterward, cells were washed with DPBS (14190–094, Gibco) 3 times
and the pellet was harvested for cell lysis. For cell lysis a buffer
containing 50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40,
2.5 mM MgCl
2
, 0.8 U/µl RNase Inhibitor (N2611, Promega), and
protease inhibitor cocktail (4693132001, Roche) was used.
Endogenous immunopurification was done as described above and
after the separation by SDS-PAGE followed by a coomassie staining
the gels were dried and analyzed by autoradiography.
2.11. RNA-isolation and quantitative RT-PCR (qPCR)
Total RNA was collected and the mRNAs were reverse transcribed
as described in [23] and [26]. Quantitative RT-PCR analysis was
performed by using Precision 2 × real-time PCR MasterMix with
SYBR green (Primerdesign, UK) using LightCycler 1.5 (Roche). For
quantification of the spliced and unspliced mRNA species the fol-
lowing primer pairs were used: spliced: #3210 (5′-TGAGGAGGCTTT
TTTGGAGG) and #3211 (5′-TTCACTAATCGAATGGATCTGTC), un-
spliced: #3210 and #640 (5′-CAATACTACTTCTTGTGGGTTGG). For
normalization, primers #1224 (5′-TCTTCCAGCCTCCCATCAGCGTT
TGG) and #1225 (5′-CAACAGAAATCCAACCTAGAGCTGCT) were used
to monitor the expression of the hGH1-mRNA of the co-transfected
pXGH5 plasmid.
3. Results
3.1. Characterization of the phosphorylation state of pICln
To investigate the phospho-status of pICln we generated a
phospho-specific pICln antibody against phosphorylated peptides of
the C-terminal region. The specificity of the phospho-pICln antibody
was tested by Western blotting, using recombinant non-phos-
phorylated pICln wildtype (wt) and the phospho-deficient mutants
193, 196, 197 A and D as negative control and S100 cytoplasm extract
as a positive control. As shown in Fig. 1A, the phospho-specific an-
tibody only recognized pICln from the S100 extract and not the re-
combinant non-phosphorylated proteins. This is in clear contrast to
a pan-pICln antibody. To verify the specificity of the phospho-spe-
cific antibody concerning phosphorylation sites within the C-ter-
minus of pICln at the serines 193, 195, and 197, recombinant wt pICln
and the two phospho-deficient mutants 193, 195, 197 A and 193, 195,
197D were subjected to a phosphorylation experiment with active
ULK1 and non-radioactive ATP. As shown in Fig. 1B, the phospho-
specific antibody recognizes only recombinant wt protein that is
phosphorylated. The two pICln mutants, which cannot be phos-
phorylated by ULK1 in the C-terminus, gave no signal with the
phospho-specific antibody (Fig. 1B).
To analyze endogenous pICln and the phospho-content of pICln,
HEK293T wild-type cell lysate was separated by size exclusion
chromatography, following immunoblot analysis using pan-specific
and phospho-specific antibodies against pICln. Total pICln (re-
presented in green color) is distributed in a wide range, whereas
pICln, phosphorylated in its C-terminus (represented in red) is only
detectable in the distinct molecular weight range from 400 kDa to
100 kDa, including the 6 S complex, the size of 158 kDa (Fig. 1C). In
contrast, treatment of HEK293T wild-type cells with the inhibitor
MRT67307 and the knockdown of ULK1 with siRNA (SD 1 G) resulted
in a marked decrease in the intensity of the phosphoantibody signal
in Western blot analyzes after size-exclusion chromatography
(Fig. 1D-F). This observation indicates that phosphorylated en-
dogenous pICln is part of the 6 S complex. Since we already have
shown in previous work [20], that ULK1 is responsible for the spe-
cific phosphorylation of pICln within its C-terminus, we investigated
the influence of ULK1 activity on the integrity of the 6 S complex.
L.M. Esser, K. Schmitz, F. Hillebrand et al. Computational and Structural Biotechnology Journal 21 (2023) 2100–2109
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L.M. Esser, K. Schmitz, F. Hillebrand et al. Computational and Structural Biotechnology Journal 21 (2023) 2100–2109
2103
To test this scenario, we performed size exclusion chromato-
graphy with lysates of HEK293T cells, cultivated in the absence or
presence of the inhibitor MRT67307 and detected the respective Sm
proteins by immunoblotting. We observed an accumulation of
SmD1/D2/E/F/G within the 6 S complex upon ULK1 inhibition
(Fig. 1G, H; red box). On the one hand, these data show the com-
position and distribution of the entire endogenous 6 S for the first
time. On the other hand, the data confirm that phosphorylation of
pICln by ULK1 reduces the amount of the intracellular 6 S inter-
mediate (Fig. 1G).
Using immunofluorescence analyses with the newly generated
phospho-specific antibody against pICln and the ULK1-specific an-
tibody, we next assessed the intracellular distribution and the in-
teractions of pICln, phospho-pICln, and ULK1. Both total pICln and
phospho-pICln co-localize with ULK1 predominantly in the cyto-
plasm. Total pICln signal intensity is significantly higher, compared
to that of phospho-pICln (Fig. 2A). Knockdown of ULK1 by siRNA has
no effect on the signal intensity of total pICln, nor has the inhibition
of kinase activity by ULK-specific kinase inhibitor MRT67307
(Fig. 2B). However, the signal intensity of phosphorylated pICln
significantly decreases upon both, inhibitor treatment and ULK1-
knockdown (Fig. 2C). Relative to total pICln, there is a 40% reduction
in phospho-pICln upon ULK1-knockdown and a 50% reduction after
inhibitor treatment (Fig. 2D). This demonstrated, that in the cell, the
phospho-status of pICln is directly regulated by ULK1.
Since we have previously shown [20], that the C-terminal phos-
phorylation of pICln by ULK1 regulates the binding properties of
pICln to Sm proteins and thus snRNP biogenesis, we asked, whether
phosphorylation of pICln affects the transfer efficiency of Sm pro-
teins to the SMN complex.
To answer this question, we assessed the pICln-mediated transfer
of
35
S-labelled reconstituted human 6 S complex onto im-
munopurified human SMN complex. Indeed, the 6 S complex suf-
ficed for the transfer of the Sm proteins to the SMN complex.
However, after treatment of the 6 S complex with recombinant ac-
tive GST-ULK1 and ATP, the transfer rate of the Sm proteins is
strongly enhanced (Fig. 3A and Supplemental Data (SD) 2 A). This
finding indicated that ULK1-dependent phosphorylation of pICln
enables a more efficient transfer of Sm proteins onto the SMN
complex and is less involved in the turnover or degradation of pICln.
Because the SMN complex has a crucial role in the late assembly
state of UsnRNPs we tested the consequence of ULK1-mediated
phosphorylation of pICln to the late assembly machinery in vivo. To
this end we performed metabolic labelling studies with [
35
S] me-
thionine in constitutive ULK1/ULK2-double knockout (DKO) MEF
cells, individually reconstituting ULK1 or ULK2 only (Fig. 3B-E; SD
2B; C). Immunopurifications using an antibody specifically re-
cognizing the m
3
G/m
7
G-cap of the snRNA, revealed that the Sm
protein transfer onto the UsnRNA is dramatically reduced in ULK1
knockout cells in comparison to the corresponding vector control
(Fig. 3D). Only MEF cells, reconstituted with ULK1 are capable to
transfer Sm proteins onto UsnRNA to the same extent as the used
control cells (Fig. 3D). This data confirms the crucial role of ULK1, but
not the closely related kinase ULK2, in the UsnRNP assembly in vivo.
3.2. ULK1 regulates splice activity via enhanced snRNP biogenesis
To further investigate the influence of pICln phosphorylation on
UsnRNP biogenesis in vivo, we analyzed the influence of ULK1 on
subsequent U1 spliceosomal activity. Therefore, we pre-incubated
HEK293T cells with the inhibitor MRT67307 and transfected them
with the previously described splicing-reporter constructs [21,22].
These reporter constructs contain either a splice donor (D1) which is
recognized by the endogenous U1 snRNA or the mutant splice donor
site 3 U that requires co-expression of the complementary U1 6 A
snRNA. Thus, co-transfection of the 3 U reporter and the U1 6 A
snRNA expression vector allows for exclusive detection of the spli-
cing activity which is dependent only on newly synthesized
UsnRNPs (Fig. 4A).
To monitor the influence of ULK inhibition on the splicing of this
specific reporter-construct derived RNAs, we performed qPCR ana-
lysis to determine the relative amount of spliced and unspliced
mRNA of the respective constructs and calculated the spliced/un-
spliced mRNA ratio.
As expected, in the absence of the ULK inhibitor we observed
predominantly spliced mRNA for both reporter constructs (90.9% for
the D1 reporter and 86.9% for the 3 U reporter, when the U1 6 A
snRNA was co-expressed) and a relatively small amount of unspliced
mRNA (9.1% for the D1 and 13.1% for the 3 U reporter, when the U1
6 A snRNA was co-expressed) (Fig. 4B). In the absence of U1 6 A
snRNA, we mainly detected unspliced mRNA when using the 3 U
reporter (8.5% of spliced and 91.5% of unspliced mRNA) (Fig. 4B),
demonstrating the requirement for of the U1 6 A snRNA expression.
However, when cells were incubated with the ULK inhibitor
MRT67307, we observed a substantially reduced spliced/unspliced
ratio (Fig. 4C) with a decrease in spliced mRNA by 31.7% for the U1
6 A snRNA-dependent and 9.7% for the endogenous D1 splice site
(Fig. 4B) with a concomitant increase in unspliced mRNA (Fig. 4B).
These results clearly demonstrate that ULK inhibition reduces
UsnRNP biogenesis and subsequent spliceosome activity in vivo.
4. Discussion
The Ser/Thr kinase ULK1 acts as a functional component of the
PRMT5 complex by specific phosphorylation of pICln [20]. Here we
characterize the phosphorylation status of endogenous pICln in
depth, by using a phospho-specific antibody against the C-terminal,
ULK1-specific phosphorylation sites of pICln. This allowed us to
study the distribution of phospho-pICln at the endogenous level.
Using immunofluorescence microscopy, it becomes evident, that
phosphorylated pICln predominantly is detectable in the cytoplasm
together with ULK1, and both methods, pharmacological inhibition
or knockdown of ULK1 by siRNA led to a clear decrease in the signal
intensity of phosphorylated pICln (Fig. 2D). However, originally de-
veloped as a ULK1/2 inhibitor [27], it turned out that the MRT67307
compound also inhibits TBK1 and IKKepsilon just as efficiently
concerning kinase activities [28]. Both kinases have their main
function predominantly in the immunological context or in immune
cells [29]. Recently, however, it has been shown that TBK1/IKKep-
silon also plays a role in energy metabolism and autophagy [30,31].
So far, we cannot exclude that TBK1 or IKKepsilon may have some
Fig. 1. Validation of pICln phosphorylation in vitro and in vivo. A, S100 extract of HEK293T cells and recombinant GST, GST-pICln wt, GST-pICln S193,195,197 A, and GST-pICln
S193,195,197D were analyzed by Tris/Glycine-SDS-PAGE and western blotting using antibodies against phosphorylated pICln and total pICln. B, Recombinant GST, GST-pICln wt,
GST-pICln S193,195,197 A, and GST-pICln S193,195,197D were incubated with 500 ng active GST-ULK1 and ATP for 1 h at 30 °C. The phosphorylation status of the proteins was
analyzed by Tris/Glycine-SDS-PAGE and western blotting as described in A. C, D S100 extracts of untreated HEK293T cells or pre-treated cells (10 µM ULK inhibitor MRT67307)
were generated with a douncer and applied to a Superdex 200 increase column. Fractions were analyzed by Tris/Glycine-SDS-PAGE and immunoblotting using antibodies against
phosphospecific pICln and total pICln. E, F, HEK293T cells were transfected with 50 nM Non-targeting control pool (E) or ULK1 siRNA (F) for 72 h. The protein complexes of the
S100 extract generated with a douncer were separated in a gel filtration with a Superdex 200 increase column. Protein complexes were analyzed by Tris/Glycine-SDS-PAGE and
western blotting as described in C, D. G, H, HEK293T cells were treated with 10 µM ULK inhibitor MRT67307 for 1 h followed by S100 extraction and subsequent gel filtration using
a Superdex 200 HiLoad 16/600 column. Obtained complexes were analyzed in comparison to untreated HEK293T cells by Tris/Tricine-SDS-PAGE and western blotting using
antibodies against the Sm proteins. The 6 S complex is highlighted by the red box.
L.M. Esser, K. Schmitz, F. Hillebrand et al. Computational and Structural Biotechnology Journal 21 (2023) 2100–2109
2104
indirect influence on snRNP-biogenesis. It will be interesting in fu-
ture work to investigate the role of both kinases in snRNP-bio-
genesis.
Our results are in line with the work of Sanchez-Olena et al. [32]
and Grimmler and colleagues [33]. Both groups could independently
show, that endogenous pICln is phosphorylated and a putative ki-
nase activity can be detected on pICln immuno-purified from the
cytoplasm. This suggests that pICln interacts with a kinase in the
cytoplasm and is consequently phosphorylated.
Interestingly, the major amount of phosphorylated pICln is found
in the size range around 158 kDa, which overlaps with the 6 S
complex of Sm proteins (Fig. 1C; E). The intermediate 6 S complex
plays a key role in UsnRNP biogenesis. The work by Chari and col-
leagues suggested, that the 6 S complex may be a kinetic trap, that is
released by the phosphorylation of pICln to keep snRNP biogenesis
Fig. 2. Inhibition of ULK1 results in a decrease of pICln phosphorylation in vivo A, Colocalization of ULK1 and pICln phospho and total. Cells were fixed with 4% PFA and afterwards
permeabilized with Triton X-100 to visualize ULK1 (red) and pICln total or phosphospecific pICln (green). The DNA was stained with DAPI (blue), scale bars 10 µm. B, HEK293T cells
were treated with 30 µM ULK inhibitor MRT67307 for 5 h or transfected with 50 nM Non-targeting control pool (NT) control or ULK1 siRNA for 72 h. Cells were fixed as described
in A to visualize ULK1 (red) and pICln total (green). The DNA was stained with DAPI (blue), scale bars 10 µm. C, HEK293T cells were treated as described in B to visualize
phosphospecific pICln (red) and pICln total (green). The DNA was stained with DAPI (blue), scale bars 10 µm. D, Inhibition or knockdown of ULK1 decreases pICln phosphorylation
in vivo. Quantification of the intensity ratio between phosphospecific pICln and pICln total was made in Fiji and the diagram in Origin, the values for MRT67307 treatment are
normalized on HEK wt value, and the values for ULK1 siRNA are normalized on NT control. The significance of the values was analyzed using a Mann-Whitney U test, the p-value of
HEK wt vs. MRT67307 and NT control vs. ULK1 siRNA was below 0.005, indicating significant differences between the values.
L.M. Esser, K. Schmitz, F. Hillebrand et al. Computational and Structural Biotechnology Journal 21 (2023) 2100–2109
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(caption on next page)
L.M. Esser, K. Schmitz, F. Hillebrand et al. Computational and Structural Biotechnology Journal 21 (2023) 2100–2109
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ongoing [18]. Complementary to our previous work, in which we
showed by in vitro binding studies, that ULK1 regulates the forma-
tion of the 6 S complex by phosphorylating pICln, our results show
that pICln is largely phosphorylated in the 6 S complex.
Concordant with this data, one should expect that, when the
kinase activity of ULK1 is inhibited, there should be an enrichment of
the 6 S complex in the cell. This is indeed the case, as shown in
Fig. 1G and H. Conversely, the transfer of Sm proteins to the SMN
complex from the 6 S complex should be increased in the presence of
ULK1. This is likewise the case (Fig. 3A, B), demonstrating that ULK1
activity is responsible for the equilibrium of endogenous 6 S com-
plex-bound Sm proteins in the cytoplasm. Since snRNPs are the
building blocks of the spliceosome [1,34], the influence of ULK1
should also be reflected in the overall splice activity. We were also
Fig. 3. ULK1 phosphorylation of pICln increases Sm protein transfer and late UsnRNP biogenesis A, pICln-mediated transfer of [
35
S]-labelled reconstituted human 6 S complex
onto immunopurified human SMN complex. To test the influence of ULK1 on the Sm protein transfer, immunopurified SMN complex was incubated with active GST-ULK1 with or
without addition of ATP which increased the binding capacity of the SMN complex. B, As control of the metabolic labelling experiment, 10% of the [
35
S] methionine lysate of ULK1/
ULK2-double knockout MEF cells as well as individually reconstituted ones with Vector control. ULK1 Flag or ULK2 HA were analyzed by a Tris-Glycine SDS-PAGE and respective
western blot. C, Whole cell lysate of MEF cells was analyzed by Tris/Glycine-SDS-PAGE and western blotting using antibodies against the Sm proteins for size comparison. D,
Analysis of late assembly machinery in vivo. Therefore metabolic labelling with [
35
S] methionine in constitutive ULK1/ULK2-double knockout MEF cells, individually reconstituting
Vector control, ULK1 Flag, or ULK2 HA only was performed. After metabolic labelling immunopurification using an antibody recognizing the m
3
G/m
7
G-cap was performed. Only
MEF cells reconstituted with ULK1 are capable to transfer Sm proteins onto UsnRNA to the same extent as MEF Vector control cells.
Fig. 4. ULK1 phosphorylation of pICln increases UsnRNP biogenesis and spliceosomal activity A, The HIV-1-based splicing reporter contains a test 5‘splice site (5‘ss) with a
mutated version of viral 5′ss D4 termed „− 1G3U“. This splice site carries two nucleotide substitutions: A-to-G at position − 1 (−1 G) and A-to-U at position + 3 (+3 U) and is poorly
recognized by the endogenous U1 snRNA due to a mismatch at position + 3. However, splice site recognition is efficiently restored following the co-expression of a U1 snRNA with
a compensatory U-to-A nucleotide exchange at position + 6, indicating that the modified U1 is assembled into functional snRNP particles within the cell. Uppercase letters within
the splice site sequences represent complementary residues, while lowercase letters represent mismatches to the U1. Mutations are highlighted in red. Base-pairing at position + 3
is highlighted by a light red box and increased font size. B; C, HEK293T cells were treated with 30 µM inhibitor MRT67307 and transiently transfected with different splicing
reporter constructs (see methods section for more details). After 20 h of inhibitor treatment cells were harvested to perform RNA isolation. Quantitative RT-PCR was executed and
mRNA was analyzed by assessing the respective ratio of spliced/unspliced form, the p value was below 0.005 (B). The equal averages including differences between inhibitor-
treated and untreated spliced and unspliced forms are listed in C (n = 3).
L.M. Esser, K. Schmitz, F. Hillebrand et al. Computational and Structural Biotechnology Journal 21 (2023) 2100–2109
2107
able to determine this by utilizing a splicing reporter assay (Fig. 4). In
summary, it can be concluded that the interaction between ULK1
and pICln regulates snRNP biogenesis and consequently also influ-
ences subsequent splicing activity in vivo.
The crucial role of pICln in neurodegenerative diseases [35] and
the unexpected link to ULK1, a key regulator of neurodifferentiation
and axonal elongation in C. elegans, mice, and humans [36–38], make
both, pICln as well as ULK1 to a new relevant target for further
studies and potential therapies of SMA and other (motor) neuronal
diseases. The successful introduction of antisense oligonucleotides
(AONs) such as nusinersen [39] or gene replacement therapy [40],
has dramatically increased the prospects for successful treatment of
SMA patients. These novel treatments focus exclusively on in-
creasing the protein levels of SMN. The long-term consequences
caused by SMN overexpression are as yet unclear [41]. A simulta-
neous increase in snRNP biogenesis via activation of ULK1 could
represent a new additional target for successful therapy. Much work
will be necessary to gather more experimental data on this – but the
association of pICln with ULK1 gives a new starting option on this so
far blind alley to answer the question of why general spliceosomal
defects predominantly do lead to neuronal manifestations.
Funding
This work was supported by grants from Deutsche
Forschungsgemeinschaft [RTG 2158 (to B.S. and S.W.), RTG 2578 (to
B.S. and S.W.), and STO 864/4-3 (to B.S.)] and the Düsseldorf School
of Oncology (funded by the Comprehensive Cancer Center
Düsseldorf/Deutsche Krebshilfe and the Medical Faculty of the
Heinrich Heine University Düsseldorf; to B.S. and S.W.).
CRediT authorship contribution statement
L.E. performed in vivo labelling experiments as well as the
Western blots of S100 extract of MEF cells, Western Blot analysis of
recombinant pICln and pre-phosphorylated recombinant pICln, size
exclusion with or without inhibitor-/ siRNA-treatment using the
phospho-specific pICln antibody, immunofluorescence analysis and
in vitro assembly assays; Ka.Sc. performed the gel filtrations with
and without inhibitor treatment, and in vitro assembly assays; S.E.,
H.F., and H.S. performed and interpreted splicing analysis; B.S. and
S.W. discussed the results; M.G. and C.P. designed the experiments
and supervised the project; all authors contributed to the writing of
the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing fi-
nancial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgments
We gratefully acknowledge Maximilian Jüdt, Qiaoping Li, David
Schlütermann, Seda Akgün and Maria Jose Mendiburo for valuable
discussions and technical support.
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.csbj.2023.03.015.
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