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Displacement of scaRNPs from CBs in Nopp140 KD cells is not caused by CRISPR/ Cas9 off-target effects. (A) Double IF demonstrates that reexpression of Nopp140 in KD cells (arrows) rescues NAP57 localization to CBs unlike in untransfected cells (asterisks). (B) Equally, Nopp140 transfection in KD cells (arrows) rescues SCARNA5/U87 localization (observed by RNA FISH) to CBs (identified by coilin double IF) unlike in untransfected cells (asterisks). (C) Transfected GFP-Nopp140 (fluorescence, arrows) rescues WDR79 and NAP57 localization to CBs unlike in untransfected cells (asterisks). (D) KD2 cells transfected with GFP-WDR79 (arrows) also rescue NAP57 localization to CBs (small arrows) but not that of residual Nopp140. (E) Transient Nopp140 KD in HeLa cells (siNopp140) shows a loss of NAP57 from CBs in siRNA transfected cells (arrows) but not from CBs of untransfected cells (small arrows). (F) Similarly, transient KD of WDR79 (si) reduced the WDR79 signal in CBs (small arrows) compared with untransfected cells (control), which was accompanied by a loss of NAP57 from CBs but not nucleoli, as previously noted (Zhong et al., 2011). (G) Transient KD of Nopp140 and WDR79, but not that of NAP57 and tubulin, was confirmed by WB on whole cell extracts. Magnification, bars = 10 µm.
Source publication
Cajal bodies (CBs) are nuclear organelles concentrating two kinds of RNA-protein complexes (RNPs), spliceosomal small nuclear (sn) and small CB-specific (sca)RNPs. Whereas the CB marker protein coilin is responsible for retaining snRNPs, the tether for scaRNPs is not known. Here we show that Nopp140, an intrinsically disordered CB phosphoprotein, i...
Contexts in source publication
Context 1
... we expressed human Nopp140 from the low-expressing UBC promoter. Over time, exogenously expressed Nopp140 first accumulated in CBs and subsequently in nucleoli of the Nopp140 KD cells (Figure 4, A-C, first panels). Unlike in untransfected cells (stars), endogenous NAP57 and SCARNA5/U87 followed the transfected Nopp140 back into CBs ( Figure 4, A and B, arrows). ...
Context 2
... time, exogenously expressed Nopp140 first accumulated in CBs and subsequently in nucleoli of the Nopp140 KD cells (Figure 4, A-C, first panels). Unlike in untransfected cells (stars), endogenous NAP57 and SCARNA5/U87 followed the transfected Nopp140 back into CBs ( Figure 4, A and B, arrows). Expression of GFP-tagged Nopp140 also restored CB localization of NAP57 and WDR79 ( Figure 4C, arrows). ...
Context 3
... in untransfected cells (stars), endogenous NAP57 and SCARNA5/U87 followed the transfected Nopp140 back into CBs ( Figure 4, A and B, arrows). Expression of GFP-tagged Nopp140 also restored CB localization of NAP57 and WDR79 ( Figure 4C, arrows). Similar results were obtained with fibrillarin, NHP2, and GAR 1 (unpublished data) and with SCARNA19/hTR and U93 (Supplemental Figure S3). ...
Context 4
... we tested whether overexpression of WDR79 in the Nopp140 KD cells might also restore scaRNP localization to CBs. Indeed, expression of GFP-tagged WDR79 in Nopp140 KD cells (arrows), restored NAP57 (as a marker for scaRNPs) to CBs ( Figure 4D, small arrows). In contrast, residual Nopp140 in the KD cells failed to reaccumulate in CBs ( Figure 4D). ...
Context 5
... expression of GFP-tagged WDR79 in Nopp140 KD cells (arrows), restored NAP57 (as a marker for scaRNPs) to CBs ( Figure 4D, small arrows). In contrast, residual Nopp140 in the KD cells failed to reaccumulate in CBs ( Figure 4D). Together with the fact that the Nopp140 loss from CBs is accompanied by a loss of WDR79 ( Figure 2G), which is restored upon Nopp140 reexpression ( Figure 4C), these data indicate that Nopp140 acts upstream of WDR79 in scaRNP localization to CBs. ...
Context 6
... contrast, residual Nopp140 in the KD cells failed to reaccumulate in CBs ( Figure 4D). Together with the fact that the Nopp140 loss from CBs is accompanied by a loss of WDR79 ( Figure 2G), which is restored upon Nopp140 reexpression ( Figure 4C), these data indicate that Nopp140 acts upstream of WDR79 in scaRNP localization to CBs. ...
Context 7
... KD of TDP43 displaced some of these scaRNAs from CBs. A loss of TDP43 from CBs in the Nopp140 KD cells suggests that Nopp140 is also responsible for the TDP43-mediated localization of scaRNPs to CBs (Supplemental Figure S4). Finally, TGS1, the trimethyl transferase responsible for hypermethylating the monomethyl caps of snRNAs in the cytoplasm and hTR and snoRNA U3 possibly in CBs, is also concentrated in CBs (Verheggen et al., 2002) (Supplemental Figure S5). ...
Context 8
... further cement the role of Nopp140 in scaRNP localization to CBs, we replicated the effects of our stable Nopp140 KD cell lines by transient KD of Nopp140 using small interfering RNAs (siRNAs). Indeed, in regular HeLa cells transfected with Nopp140 siRNAs ( Figure 4E, arrows), NAP57 was lost from CBs but not nucleoli and in contrast to CBs of untransfected cells ( Figure 4E, small arrows). Similar results were observed in U2OS cells showing this to be a more general effect not unique to HeLa cells (unpublished data). ...
Context 9
... further cement the role of Nopp140 in scaRNP localization to CBs, we replicated the effects of our stable Nopp140 KD cell lines by transient KD of Nopp140 using small interfering RNAs (siRNAs). Indeed, in regular HeLa cells transfected with Nopp140 siRNAs ( Figure 4E, arrows), NAP57 was lost from CBs but not nucleoli and in contrast to CBs of untransfected cells ( Figure 4E, small arrows). Similar results were observed in U2OS cells showing this to be a more general effect not unique to HeLa cells (unpublished data). ...
Context 10
... results were observed in U2OS cells showing this to be a more general effect not unique to HeLa cells (unpublished data). Finally, we replicated the previously reported effect of transient WDR79 KD (Zhong et al., 2011) documenting a specific loss of NAP57 from CBs but not nucleoli in cells transfected with WDR79 siRNA ( Figure 4F). In both cases, KD was confirmed by WB of whole cell extracts ( Figure 4G). ...
Context 11
... we replicated the previously reported effect of transient WDR79 KD (Zhong et al., 2011) documenting a specific loss of NAP57 from CBs but not nucleoli in cells transfected with WDR79 siRNA ( Figure 4F). In both cases, KD was confirmed by WB of whole cell extracts ( Figure 4G). Overall, we conclude that Nopp140 is responsible for scaRNP accumulation in CBs upstream of WDR79. ...
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Dyskeratosis congenita is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for bone marrow failure in dyskeratosis congenita patients is allogeneic hematopoietic stem cell transplantation. However due to the toxicity associated to allogen...
Dyskerin is a protein involved in the formation of small nucleolar and small Cajal body ribonucleoproteins. These complexes participate in RNA pseudouridylation and are also components of the telomerase complex required for telomere elongation. Dyskerin mutations cause a rare disease, X-linked dyskeratosis congenita, with no curative treatment. The...
Citations
... From the top UGOTs for the combined D123 domains, it is clear, though, that abundant RNA processing in the nuclear Cajal body that occurs in the Li ceases or drastically decreases once cells migrate to the Pe. The Cajal body function may be associated with other UGOTs, including regulation of telomere maintenance via telomerase, since the enzyme mRNA has been found to associate with the Cajal body and its telomere length regulation [34,35]. Other intriguing UGOTs, potentially reflecting the much less differentiated state of the D123 domain in comparison to D4i.1 are the regulation of hematopoietic progenitor cell differentiation, the regulation of stem cell differentiation, and the regulation of stem cell population maintenance (all these terms are present in both the D0 and D1, and thus do not show up in either the D0 or D1 Table 8 UGOTs). ...
The corneal epithelium (CE) is spread between two domains, the outer vascularized limbus and the avascular cornea proper. Epithelial cells undergo constant migration from the limbus to the vision-critical central cornea. Coordinated with this migration, the cells undergo differentiation changes where a pool of unique stem/precursor cells at the limbus yields the mature cells that reach the corneal center. Differentiation is heralded by the expression of the corneal-specific Krt12. Processing data acquired by scRNA-Seq showed that the increase in Krt12 expression occurs in four distinct steps within the limbus, plus a single continuous increase in the cornea. Differential gene analysis demonstrated that these domains reflect discreet stages of CE differentiation and yielded extensive information of the genes undergoing down- or upregulation in the sequential transition from less to more differentiate conditions. The approach allowed the identification of multiple gene cohorts, including (a) the genes which have maximal expression in the most primitive, Krt12-negative cell cohort, which is likely to include the stem/precursor cells; (b) the sets of genes that undergo continuous increase or decrease along the whole differentiation path; and (c) the genes showing maximal positive or negative correlation with the changes in Krt12.
... From the top UGOTs for the combine D123 domains, it is clear, though, that abundant RNA processing in the nuclear Cajal body that occurs in the Li, ceases or drastically decreases once cells migrate to the Pe. The Cajal body function may be associated the UGOT, including regulation of telomere maintenance via telomerases, since the enzyme mRNA has been found to associate with the Cajal body and its telomere length regulation [19,20]. Other intriguing UGOTs, potentially reflecting the much less differentiated state of the D123 domain in comparison to D4i.1 are, regulation of hematopoietic progenitor cell differentiation, the regulation of stem cell differentiation and regulation of stem cell population maintenance. ...
... At least for a specific transformed cell NR2F2 had been shown to act as a promoter of stemness [17,18]. ID3 is a repressor of basic helix-loop-helix transcription factors and had been shown to support human embryonic stem cell maintenance [20]. Both are particularly interesting subjects for further research. ...
The corneal epithelium (CE) is spread between two domains, the outer vascularized limbus and the avascular cornea proper. Epithelial cells undergo constant migration from the limbus to the vision-critical central cornea. Coordinated with this migration the cells undergo differentiation changes where a pool of unique stem/precursor cells at the limbus yields the mature cells that reach the corneal center. Differentiation is heralded by the expression of the corneal-specific Krt12. Processing data acquired by scRNA-Seq we found that the increase of Krt12 expression occurs in four distinct steps within the limbus plus a single continuous increase in the cornea. Differential gene analysis demonstrated that these domains reflect discreet stages of CE differentiation and yielded extensive information of the genes undergoing down or up regulation in the sequential transition from less to more differentiate conditions. The approach allowed the identification of multiple gene cohorts, including a) the genes that have maximal expression in the most primitive, Krt12-negative cell cohort, which is likely to include the stem/precursor cells; b) the sets of genes that undergo continuous increase or decrease along the whole differentiation path, and c) and the genes showing maximal positive or negative correlation with the changes in Krt12.
... Telomerase is formed by TERT, telomerase RNA gene (TERC) and some proteins related to telomere enzyme activity and assembly [9,10]. TCAB1 (also known as WRAP53 or WDR79) is a key core component of telomerase protein discovered in 2009, which mainly exists in Cajal body [11], can transport telomerase from the Cajal body to the position of telomere synthesis at the end of telomere, and thereby ensure the normal activation mechanism to maintain the telomere length [12]. TCAB1 belongs to WD40 family homologue. ...
Objectives
Telomerase Cajal body protein 1 (TCAB1) is closely related to the occurrence, development and prognosis of tumors, and may affect the sensitivity of tumor radiotherapy. There are no reports about the effect of TCAB1 gene expression on proliferation and apoptosis of HEPG2 cells. We plan to investigate whether silencing TCAB1 using siRNA is helpful for the diagnosis and treatment of hepatocellular carcinoma.
Methods
Three siRNA sequences (siTCAB1-1, siTCAB1-2, siTCAB1-3) targeting TCAB1 gene and one negative sequence (NC) were designed, synthesized and then transfected into HEPG2 cells, separately. The expressions of TCAB1 and telomerase reverse transcriptase (TERT) in mRNA and protein level were detected by RT-qPCR and Western blot assays. Moreover, proliferation and apoptosis of HEPG2 cells were measured by MTT assay, and flow cytometry, respectively.
Results
RT-qPCR and Western blot data both showed that TCAB1 expression in the siTCAB1 group was significantly lower than that in the blank control and NC groups (p < 0.05). However, TERT expression was not significantly different among those groups (p > 0.05). MTT result showed that HEPG2 cells proliferation in the siTCAB1 group was lower than that in the blank control and NC groups (p < 0.05). The apoptotic rate in the siTCAB1 group was significantly increased compared with the blank control and NC groups (p < 0.01).
Conclusions
Silencing TCAB1 can inhibit proliferation and promote apoptosis of HEPG2 cells, providing a potential therapeutic method for diagnosis and treatment of hepatocellular carcinoma.
... Nopp140 is required to recruit and retain all scaRNPs in Cajal bodies. Nopp140 plays an important role in the formation of Cajal bodies and the localization of scaRNAs and the telomerase RNA within them [227]. Nopp140 may also function as a transport factor between the nucleolus and Cajal bodies [228]. ...
... Even the earlier study supported the notion that TCAB1 can localize to telomeres in Cajal body-independent manner [280]. Surprisingly, the absence of telomerase in Cajal bodies due to the knockdown of Nopp140 leads to gradual telomere elongation in cancer cells [227]. Moreover, recent imaging data obtained from living cells have shown that no more than 10% of hTR is localized in Cajal bodies; the rest is distributed throughout the nucleoplasm [219]. ...
Telomere length is associated with the proliferative potential of cells. Telomerase is an enzyme that elongates telomeres throughout the entire lifespan of an organism in stem cells, germ cells, and cells of constantly renewed tissues. It is activated during cellular division, including regeneration and immune responses. The biogenesis of telomerase components and their assembly and functional localization to the telomere is a complex system regulated at multiple levels, where each step must be tuned to the cellular requirements. Any defect in the function or localization of the components of the telomerase biogenesis and functional system will affect the maintenance of telomere length, which is critical to the processes of regeneration, immune response, embryonic development, and cancer progression. An understanding of the regulatory mechanisms of telomerase biogenesis and activity is necessary for the development of approaches toward manipulating telomerase to influence these processes. The present review focuses on the molecular mechanisms involved in the major steps of telomerase regulation and the role of post-transcriptional and post-translational modifications in telomerase biogenesis and function in yeast and vertebrates.
... The retention of H/ACA scaRNPs and telomerase in CBs requires TCAB1 (WDR79) that specifically interacts with the CAB sequence present within these RNAs (43)(44)(45)(46). Nopp140 is also important to recruit and retain H/ACA scaRNPs in CBs (47) and is believed to function in H/ACA snoRNP traffic from CBs to nucleoli (48,49). Other factors are for a long time thought to participate in H/ACA RNP biogenesis but their roles are still enigmatic. ...
The conserved H/ACA RNPs consist of one H/ACA RNA and 4 core proteins: dyskerin, NHP2, NOP10, and GAR1. Its assembly requires several assembly factors. A pre-particle containing the nascent RNAs, dyskerin, NOP10, NHP2 and NAF1 is assembled co-transcriptionally. NAF1 is later replaced by GAR1 to form mature RNPs. In this study, we explore the mechanism leading to the assembly of H/ACA RNPs. We performed the analysis of GAR1, NHP2, SHQ1 and NAF1 proteomes by quantitative SILAC proteomic, and analyzed purified complexes containing these proteins by sedimentation on glycerol gradient. We propose the formation of several distinct intermediate complexes during H/ACA RNP assembly, notably the formation of early protein-only complexes containing at least the core proteins dyskerin, NOP10, and NHP2, and the assembly factors SHQ1 and NAF1. We also identified new proteins associated with GAR1, NHP2, SHQ1 and NAF1, which can be important for box H/ACA assembly or function. Moreover, even though GAR1 is regulated by methylations, the nature, localization, and functions of these methylations are not well known. Our MS analysis of purified GAR1 revealed new sites of arginine methylations. Additionally, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs, even though with less efficiency than methylated ones.
... Nevertheless, a notable exception was Nopp140 ( Supplementary Fig. 1g), which did colocalize with NTD NLS . Nopp140 is a nucleolar and CB protein previously shown to interact with coilin NTD 39,40 that has been linked with the accumulation of snoRNP components and scaRNPs in CBs and residual bodies 6,17,41 . The snoRNP component, fibrillarin, was broadly nucleoplasmic and not welllocalized to the NTD NLS puncta, suggesting the NTD NLS puncta are not limited to possible residual bodies observed previously in these cells 17 . ...
... The observed colocalization of Nopp140 with nuclear NTD NLS in puncta raised the question of whether the coilin NTD-Nopp140 interactions contribute to CB assembly, which is a controversial point in the literature. Partial knockdown of Nopp140 by stable CRISPR/Cas9 editing did not disturb CB formation 6,41 , while two other studies showed that loss of Nopp140 is correlated with CB disassembly 40,42 . To determine whether Nopp140 is required for CB assembly and maintenance in our system, we depleted Nopp140 and coilin proteins from HeLa cells using siRNAs ( Fig. 2a; note that the NOLC1 gene encodes Nopp140 protein). ...
Cajal bodies (CBs) are ubiquitous nuclear membraneless organelles (MLOs) that concentrate and promote efficient biogenesis of snRNA-protein complexes involved in splicing (snRNPs). Depletion of the CB scaffolding protein coilin disperses snRNPs, making CBs a model system for studying the structure and function of MLOs. Although it is assumed that CBs form through condensation, the biomolecular interactions responsible remain elusive. Here, we discover the unexpected capacity of coilin’s N-terminal domain (NTD) to form extensive fibrils in the cytoplasm and discrete nuclear puncta in vivo. Single amino acid mutational analysis reveals distinct molecular interactions between coilin NTD proteins to form fibrils and additional NTD interactions with the nuclear Nopp140 protein to form puncta. We provide evidence that Nopp140 has condensation capacity and is required for CB assembly. From these observations, we propose a model in which coilin NTD–NTD mediated assemblies make multivalent contacts with Nopp140 to achieve biomolecular condensation in the nucleus.
... Components of the alternative lengthening of telomeres pathway (ATRX, PML and SLX4) were also among the new loci as well as genes encoding factors that post-translationally modify key telomere proteins, including UPS7, which encodes a protein that deubiquitinates both POT1 and ACD 19,20 . Genes within both known (TERC, TERT and NAF1) and new (DKC1, TEP1, SMG6, SHQ1, NOLC1 and RUVBL1) loci encode core components of proteins that regulate the assembly and activity of telomerase (Supplementary Note) [21][22][23][24] . Before telomerase assembly, TERC undergoes complex processing 25 . ...
Telomeres, the end fragments of chromosomes, play key roles in cellular proliferation and senescence. Here we characterize the genetic architecture of naturally occurring variation in leukocyte telomere length (LTL) and identify causal links between LTL and biomedical phenotypes in 472,174 well-characterized UK Biobank participants. We identified 197 independent sentinel variants associated with LTL at 138 genomic loci (108 new). Genetically determined differences in LTL were associated with multiple biological traits, ranging from height to bone marrow function, as well as several diseases spanning neoplastic, vascular and inflammatory pathologies. Finally, we estimated that, at the age of 40 years, people with an LTL >1 s.d. shorter than the population mean had a 2.5-year-lower life expectancy compared with the group with ≥1 s.d. longer LDL. Overall, we furnish new insights into the genetic regulation of LTL, reveal wide-ranging influences of LTL on physiological traits, diseases and longevity, and provide a powerful resource available to the global research community.
... Depletion of Nopp140 in human cell lines results in structural changes in the Cajal bodies, reducing their size, along with the loss of scaRNPs, including the telomerase holoenzyme, in these membraneless compartments. Nopp140 thus plays an important function in Cajal body formation and localization of scaRNPs (Bizarro et al. 2019 (Ghanim et al. 2021). Thus, it is still not possible to confirm whether all predicted structural rearrangements during assembly and function are conserved from archaeal and yeast homologues to humans. ...
Dyskerin and its homologues are ancient and conserved enzymes that catalyse the most common posttranscriptional modification found in cells, pseudouridylation. The resulting pseudouridines provide stability to RNA molecules and regulate ribosome biogenesis and splicing events. Dyskerin does not act independently – it is the core component of a protein heterotetramer, which associates with RNAs that contain the H/ACA motif. The variety of H/ACA RNAs that guide the function of this ribonucleoprotein (RNP) complex highlight the diversity of cellular processes in which dyskerin participates. When associated with small nucleolar (sno) RNAs, it regulates ribosomal (r) RNAs and ribosome biogenesis. By interacting with small Cajal Body (sca) RNAs, it targets small nuclear (sn) RNAs to regulate pre-mRNA splicing. As a component of the telomerase holoenzyme, dyskerin binds to the telomerase RNA to modulate telomere maintenance. In a disease context, dyskerin malfunction can result in multiple detrimental phenotypes. Mutations in DKC1, the gene that encodes dyskerin, cause the premature aging syndrome X-linked dyskeratosis congenita (X-DC), a still incurable disorder that typically leads to bone marrow failure. In this review, we present the classical and most recent findings on this essential protein, discussing the evolutionary, structural and functional aspects of dyskerin and the H/ACA RNP. The latest research underscores the role that dyskerin plays in the regulation of gene expression, translation efficiency and telomere maintenance, along with the impacts that defective dyskerin has on aging, cell proliferation, haematopoietic potential and cancer.
... High-resolution CRISPR screens identified Nopp140 as essential for cell survival (Hart et al. 2015;Wang et al. 2015). Using a targeted CRISPR/Cas9 approach in polyploid HeLa cells, we established three cell lines with very low levels of Nopp140 (∼1%-7% residual protein level); i.e., Nopp140 knockdown (KD) cell lines (Bizarro et al. 2019). Surprisingly, Nopp140 KD cells do not exhibit any growth or gross phenotypes. ...
... Nevertheless, the KD cells reveal subtle but clear differences in Nopp140 chaperoned activities filtering nonessential from essential functions. We showed that one of these nonessential functions is corralling scaRNPs in Cajal bodies (Bizarro et al. 2019). In Nopp140 low-expressing cells, all scaRNPs are released from Cajal bodies but the overall levels and integrity of the RNPs remain unaffected. ...
... In Nopp140 low-expressing cells, all scaRNPs are released from Cajal bodies but the overall levels and integrity of the RNPs remain unaffected. As one of the consequences, the telomerase scaRNP is no longer sheltered in CBs but has continuous access to telomeres extending them gradually (Bizarro et al. 2019). Here we present the consequences of Nopp140 KD for all other scaRNPs when no longer maintained in CBs and for snoRNPs in nucleoli. ...
Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB)-specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at ∼80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2'-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.
... Together with the repopulation of CBs with scaRNPs in the rescue cells, this data further indicates that scaRNPs normally reside in the granules of CBs. We previously reported the same effects of Nopp140 KD on nucleoli and CBs in other KD cells, KD1a, demonstrating that this is not a clonal aberration (Bizarro et al., 2019). ...
... In the absence of the CK2 inhibitor, transfection of Nopp140 (cells with arrows) caused both proteins to localize to CBs and nucleoli. However, in the residual untransfected Nopp140 KD cells (asterisks), NAP57 localized only to nucleoli but not CBs (Fig. 2D, upper panels) consistent with our previous results (Bizarro et al., 2019). In the presence of newly translated,unphosphorylated Nopp140 similarly accumulated only in nucleoli but not CBs (Fig. 2D,lower panels,arrow) demonstrating that Nopp140 phosphorylation was required for CB targeting. ...
... In the presence of newly translated,unphosphorylated Nopp140 similarly accumulated only in nucleoli but not CBs (Fig. 2D,lower panels,arrow) demonstrating that Nopp140 phosphorylation was required for CB targeting. Consistent with the fact that NAP57 accumulation in CBs depends on the localization of Nopp140 in CBs (Bizarro et al., 2019), NAP57 stayed in nucleoli but was excluded from CBs in the presence of CX-4945, even in Nopp140 transfected cells (Fig. 2D, lower panels, arrow). In summary, CK2 phosphorylation of Nopp140 is required for the accumulation of both proteins in CBs and by extension for that of scaRNPs. ...
Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB) specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at some 80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2′-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.
