The Cajal body (CB) is a conserved non-membrane nuclear structure where several steps of small nuclear RNP particle (snRNP) biogenesis take place. It has been proposed that CB formation follows a liquid-liquid phase separation model, but this hypothesis has never been rigorously tested. Here, we applied live-cell imaging to show that the key CB assembly factor coilin is mobile within the CB, and we revealed a diffusion barrier that limits the coilin exchange between CBs and the nucleoplasm. We generated single aa mutations and demonstrated that RNA-dependent coilin oligomerization and coilin interaction with snRNP are essential for CB formation and maintenance. We applied these data to formulate a mathematical model that links the movement of coilin within the nucleoplasm, CB, and across the boundary with its oligomerization and snRNP binding. Our results illustrate CB as a structure dynamically responding to snRNP assembly and recycling.

• MeSH
• Coiled Bodies * metabolism   genetics   MeSH
• HeLa Cells MeSH
• Nuclear Proteins * metabolism   genetics   MeSH
• Humans MeSH
• Protein Multimerization MeSH
• Mutation MeSH
• Ribonucleoproteins, Small Nuclear * metabolism   genetics   MeSH
• Spliceosomes * metabolism   genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Nuclear Proteins * MeSH
• p80-coilin MeSH Browser
• Ribonucleoproteins, Small Nuclear * MeSH

Spliceosomal snRNPs are multicomponent particles that undergo a complex maturation pathway. Human Sm-class snRNAs are generated as 3'-end extended precursors, which are exported to the cytoplasm and assembled together with Sm proteins into core RNPs by the SMN complex. Here, we provide evidence that these pre-snRNA substrates contain compact, evolutionarily conserved secondary structures that overlap with the Sm binding site. These structural motifs in pre-snRNAs are predicted to interfere with Sm core assembly. We model structural rearrangements that lead to an open pre-snRNA conformation compatible with Sm protein interaction. The predicted rearrangement pathway is conserved in Metazoa and requires an external factor that initiates snRNA remodeling. We show that the essential helicase Gemin3, which is a component of the SMN complex, is crucial for snRNA structural rearrangements during snRNP maturation. The SMN complex thus facilitates ATP-driven structural changes in snRNAs that expose the Sm site and enable Sm protein binding.

• MeSH
• HeLa Cells MeSH
• snRNP Core Proteins genetics   MeSH
• Humans MeSH
• RNA Precursors * metabolism   MeSH
• SMN Complex Proteins metabolism   MeSH
• Ribonucleoproteins, Small Nuclear metabolism   MeSH
• RNA, Small Nuclear * metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• snRNP Core Proteins MeSH
• RNA Precursors * MeSH
• SMN Complex Proteins MeSH
• Ribonucleoproteins, Small Nuclear MeSH
• RNA, Small Nuclear * MeSH

Mutations in BRAT1, encoding BRCA1-associated ATM activator 1, have been associated with neurodevelopmental and neurodegenerative disorders characterized by heterogeneous phenotypes with varying levels of clinical severity. However, the underlying molecular mechanisms of disease pathology remain poorly understood. Here, we show that BRAT1 tightly interacts with INTS9/INTS11 subunits of the Integrator complex that processes 3' ends of various noncoding RNAs and pre-mRNAs. We find that Integrator functions are disrupted by BRAT1 deletion. In particular, defects in BRAT1 impede proper 3' end processing of UsnRNAs and snoRNAs, replication-dependent histone pre-mRNA processing, and alter the expression of protein-coding genes. Importantly, impairments in Integrator function are also evident in patient-derived cells from BRAT1 related neurological disease. Collectively, our data suggest that defects in BRAT1 interfere with proper Integrator functions, leading to incorrect expression of RNAs and proteins, resulting in neurodegeneration.

• MeSH
• Phenotype MeSH
• Histones MeSH
• Nuclear Proteins * genetics   MeSH
• Humans MeSH
• Mutation MeSH
• Neurodegenerative Diseases * genetics   MeSH
• RNA Processing, Post-Transcriptional * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BRAT1 protein, human MeSH Browser
• Histones MeSH
• Nuclear Proteins * MeSH

Coilin is a conserved protein essential for integrity of nuclear membrane-less inclusions called Cajal bodies. Here, we report an amino acid substitution (p.K496E) found in a widely-used human EGFP-coilin construct that has a dominant-negative effect on Cajal body formation. We show that this coilin-K496E variant fails to rescue Cajal bodies in cells lacking endogenous coilin, whereas the wild-type construct restores Cajal bodies in mouse and human coilin-knockout cells. In cells containing endogenous coilin, both the wild-type and K496E variant proteins accumulate in Cajal bodies. However, high-level overexpression of coilin-K496E causes Cajal body disintegration. Thus, a mutation in the C-terminal region of human coilin can disrupt Cajal body assembly. Caution should be used when interpreting data from coilin plasmids that are derived from this variant (currently deposited at Addgene).

• Keywords
• Cajal bodies, Coilin, Mutation,
• MeSH
• Point Mutation * genetics   MeSH
• Coiled Bodies * genetics   MeSH
• HeLa Cells MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• Mutation genetics   MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Nuclear Proteins MeSH

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) undergo a complex maturation pathway containing multiple steps in the nucleus and in the cytoplasm. snRNP biogenesis is strictly proofread and several quality control checkpoints are placed along the pathway. Here, we analyzed the fate of small nuclear RNAs (snRNAs) that are unable to acquire a ring of Sm proteins. We showed that snRNAs lacking the Sm ring are unstable and accumulate in P-bodies in an LSm1-dependent manner. We further provide evidence that defective snRNAs without the Sm binding site are uridylated at the 3' end and associate with DIS3L2 3'→5' exoribonuclease and LSm proteins. Finally, inhibition of 5'→3' exoribonuclease XRN1 increases association of ΔSm snRNAs with DIS3L2, which indicates competition and compensation between these two degradation enzymes. Together, we provide evidence that defective snRNAs without the Sm ring are uridylated and degraded by alternative pathways involving either DIS3L2 or LSm proteins and XRN1.

• MeSH
• Exoribonucleases metabolism   MeSH
• HeLa Cells MeSH
• Nucleic Acid Conformation * MeSH
• Humans MeSH
• Organelles metabolism   MeSH
• SMN Complex Proteins metabolism   MeSH
• RNA-Binding Proteins metabolism   MeSH
• Proto-Oncogene Proteins metabolism   MeSH
• RNA, Small Nuclear chemistry   metabolism   MeSH
• Base Sequence MeSH
• RNA Stability MeSH
• RNA Transport * MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DIS3L2 protein, human MeSH Browser
• Exoribonucleases MeSH
• GEMIN5 protein, human MeSH Browser
• LSM1 protein, human MeSH Browser
• SMN Complex Proteins MeSH
• RNA-Binding Proteins MeSH
• Proto-Oncogene Proteins MeSH
• RNA, Small Nuclear MeSH

Cajal bodies (CBs) are nuclear non-membrane bound organelles where small nuclear ribonucleoprotein particles (snRNPs) undergo their final maturation and quality control before they are released to the nucleoplasm. However, the molecular mechanism how immature snRNPs are targeted and retained in CBs has yet to be described. Here, we microinjected and expressed various snRNA deletion mutants as well as chimeric 7SK, Alu or bacterial SRP non-coding RNAs and provide evidence that Sm and SMN binding sites are necessary and sufficient for CB localization of snRNAs. We further show that Sm proteins, and specifically their GR-rich domains, are important for accumulating snRNPs in CBs. Accordingly, core snRNPs containing the Sm proteins, but not naked snRNAs, restore the formation of CBs after their depletion. Finally, we show that immature but not fully assembled snRNPs are able to induce CB formation and that microinjection of an excess of U2 snRNP-specific proteins, which promotes U2 snRNP maturation, chases U2 snRNA from CBs. We propose that the accessibility of the Sm ring represents the molecular basis for the quality control of the final maturation of snRNPs and the sequestration of immature particles in CBs.

• MeSH
• Cell Nucleus genetics   MeSH
• Coiled Bodies genetics   metabolism   MeSH
• HeLa Cells MeSH
• Humans MeSH
• Ribonucleoprotein, U2 Small Nuclear genetics   MeSH
• Gene Expression Regulation genetics   MeSH
• RNA, Small Nuclear genetics   MeSH
• Spliceosomes genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ribonucleoprotein, U2 Small Nuclear MeSH
• RNA, Small Nuclear MeSH
• U2 small nuclear RNA MeSH Browser

Splicing is catalyzed by the spliceosome, a complex of five major small nuclear ribonucleoprotein particles (snRNPs). The pre-mRNA splicing factor PRPF8 is a crucial component of the U5 snRNP, and together with EFTUD2 and SNRNP200, it forms a central module of the spliceosome. Using quantitative proteomics, we identified assembly intermediates containing PRPF8, EFTUD2, and SNRNP200 in association with the HSP90/R2TP complex, its ZNHIT2 cofactor, and additional proteins. HSP90 and R2TP bind unassembled U5 proteins in the cytoplasm, stabilize them, and promote the formation of the U5 snRNP. We further found that PRPF8 mutants causing Retinitis pigmentosa assemble less efficiently with the U5 snRNP and bind more strongly to R2TP, with one mutant retained in the cytoplasm in an R2TP-dependent manner. We propose that the HSP90/R2TP chaperone system promotes the assembly of a key module of U5 snRNP while assuring the quality control of PRPF8. The proteomics data further reveal new interactions between R2TP and the tuberous sclerosis complex (TSC), pointing to a potential link between growth signals and the assembly of key cellular machines.

• MeSH
• Peptide Elongation Factors genetics   metabolism   MeSH
• HeLa Cells MeSH
• Protein Interaction Domains and Motifs MeSH
• Humans MeSH
• Ribonucleoprotein, U1 Small Nuclear metabolism   MeSH
• Ribonucleoprotein, U4-U6 Small Nuclear metabolism   MeSH
• Ribonucleoprotein, U5 Small Nuclear genetics   metabolism   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Multiprotein Complexes MeSH
• Mutation MeSH
• RNA Precursors genetics   metabolism   MeSH
• HSP90 Heat-Shock Proteins metabolism   MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Calcium-Binding Proteins metabolism   MeSH
• Proteomics methods   MeSH
• Retinitis Pigmentosa genetics   metabolism   MeSH
• RNA Interference MeSH
• RNA Splicing * MeSH
• Protein Stability MeSH
• Transfection MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• EFTUD2 protein, human MeSH Browser
• Peptide Elongation Factors MeSH
• Ribonucleoprotein, U1 Small Nuclear MeSH
• Ribonucleoprotein, U4-U6 Small Nuclear MeSH
• Ribonucleoprotein, U5 Small Nuclear MeSH
• RNA, Messenger MeSH
• Multiprotein Complexes MeSH
• RNA Precursors MeSH
• HSP90 Heat-Shock Proteins MeSH
• RNA-Binding Proteins MeSH
• Calcium-Binding Proteins MeSH
• PRPF8 protein, human MeSH Browser
• TESC protein, human MeSH Browser