A Stochastic View of Spliceosome Assembly and Recycling in the Nucleus

Rino, José Pedro; Carvalho, Tereza Cristina de; Braga, Juan C.; Desterro, Joana; Lührmann, Reinhard; Carmo‐Fonseca, Maria

doi:10.1371/journal.pcbi.0030201

Cited by 58 publications

(84 citation statements)

References 58 publications

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“…Spliceosomal snRNPs explore the nuclear volume by Brownian diffusion 3,15,43 . Given this, the rate at which immature snRNPs encounter partner proteins or other snRNPs will determine how quickly their assembly occurs.…”

Section: Discussionmentioning

confidence: 99%

Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis

Strzelecka

Trowitzsch

Weber

et al. 2010

Nat Struct Mol Biol

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0 3 a r t i c l e sIn eukaryotes, organelles delimited by lipid bilayers support the organization of cellular functions, as exemplified by the segregation of chromatin into the cell nucleus. However, many subcellular compartments-nucleoli, Cajal and PML bodies, polar granules, stress granules, P bodies and others-lack membranes. How these structures contribute to cellular physiology is largely unknown 1 . The Cajal body (CB) is a 0.5-to 1-µm nuclear compartment initially described by Ramon y Cajal in silver-stained sections of vertebrate cerebral cortex 2 . Because the CB is conserved in evolution and is uniquely marked by the protein coilin, it has served as a general model for subnuclear structure and function 1,2 . Numerous factors essential for pre-mRNA splicing, histone mRNA 3′-end processing, telomere maintenance and rRNA processing are concentrated in CBs. Yet CB components exchange rapidly with the nucleoplasm, where most of these processes occur 3 . The challenge, therefore, has been to identify the function of CBs.Despite the localization of splicing machinery in CBs, they are not the sites of pre-mRNA splicing 4,5 . Splicing occurs throughout the nucleus and is catalyzed by the spliceosome, a macromolecular complex formed on pre-mRNA from five essential snRNPs 6 . Each spliceosomal snRNP comprises a unique 100-to 200-nucleotide snRNA (U1, U2, U4, U5 and U6) that is post-transcriptionally modified and associated with a number of snRNP-specific proteins 6,7 . Several roles for CBs in spliceosomal snRNP biogenesis have been proposed based on the localization of specific steps in snRNP assembly in the CBs of cultured cell lines. After transcription and export to the cytoplasm, U1, U2, U4 and U5 snRNAs receive a heteroheptameric ring of Sm proteins assembled by the SMN complex; subsequently, their 5′ ends are hypermethylated 8 . The Sm ring and trimethylguanosine (TMG) cap are signals for snRNP reimport into the nucleus, where these still-immature core snRNPs first concentrate in CBs 9-12 . CBs contain scaRNAs, which then guide site-specific modification of the snRNAs 13 . Importantly, intermediates in the final snRNP maturation steps, reflecting RNA structural rearrangements and the recruitment of snRNP-specific proteins, are highly concentrated in CBs 14-17 . These observations have led to the proposal that CBs are the sites of snRNP assembly, but an essential role for CBs in this process has not been shown.The coilin protein may hold the key to CB function. Immunoelectron microscopy studies of CBs reveal thread-like, coilin-positive aggregates, suggesting that coilin is integral to CB structure 2 . Consistent with this, coilin resides longer in CBs than any other examined component in vivo 3,18 . Notably, depletion of coilin results in the dispersal of many CB components. Without coilin, spliceosomal factors become nucleoplasmic, and other classes of CB-localized factors (for example, SMN, fibrillarin and small Cajal body-specific RNAs (scaRNAs)) continue to self-associate in discrete 'residual bodies' ...

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Section: Discussionmentioning

confidence: 99%

Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis

Strzelecka

Trowitzsch

Weber

et al. 2010

Nat Struct Mol Biol

Self Cite

150

164

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“…Surprisingly, trafficking kinetics was independent of ongoing transcription and splicing, challenging the view that spliceosomal components are stored in nuclear speckles until a signal triggers their recruitment to nascent introns; rather, it is more likely that the building blocks of the spliceosome are constantly roaming the nuclear space until they collide and transiently interact with either a nascent pre-mRNA, to form a spliceosome, or with other splicing proteins localized in nuclear speckles. 24 FRAP analysis further revealed that core spliceosomal snRNP proteins have a residence time of 15-30 s in the nucleoplasm, where spliceosomal snRNPs are thought to interact predominantly with pre-mRNA. 25 Based on these results it was suggested that splicing can be accomplished within 30 s, which is significantly more rapid than previously reported.…”

mentioning

confidence: 99%

The timing of pre-mRNA splicing visualized in real-time

Carmo‐Fonseca¹,

Kirchhausen²

2014

Nucleus

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“…59 Finally, this arrangement might also aid in the attack of BPA at the 5 0 splice site in a diffusion limited regime by limiting pre-mRNA motions in the local vicinity of the spliceosomal proteins. 112 Our model shows that SF3b components are imperative for spliceosome to achieve branch point adenosine recognition fidelity.…”

Section: Sf3b -A Fuzzy Complexmentioning

confidence: 86%

Structural and mechanistic insights into human splicing factor SF3b complex derived using an integrated approach guided by the cryo-EM density maps

et al. 2016

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Pre-mRNA splicing in eukaryotes is performed by the spliceosome, a highly complex macromolecular machine. SF3b is a multi-protein complex which recognizes the branch point adenosine of pre-mRNA as part of a larger U2 snRNP or U11/U12 di-snRNP in the dynamic spliceosome machinery. Although a cryo-EM map is available for human SF3b complex, the structure and relative spatial arrangement of all components in the complex are not yet known. We have recognized folds of domains in various proteins in the assembly and generated comparative models. Using an integrative approach involving structural and other experimental data, guided by the available cryo-EM density map, we deciphered a pseudoatomic model of the closed form of SF3b which is found to be a "fuzzy complex" with highly flexible components and multiplicity of folds. Further, the model provides structural information for 5 proteins (SF3b10, SF3b155, SF3b145, SF3b130 and SF3b14b) and localization information for 4 proteins (SF3b10, SF3b145, SF3b130 and SF3b14b) in the assembly for the first time. Integration of this model with the available U11/U12 di-snRNP cryo-EM map enabled elucidation of an open form. This now provides new insights on the mechanistic features involved in the transition between closed and open forms pivoted by a hinge region in the SF3b155 protein that also harbors cancer causing mutations. Moreover, the open form guided model of the 5 0 end of U12 snRNA, which includes the branch point duplex, shows that the architecture of SF3b acts as a scaffold for U12 snRNA: pre-mRNA branch point duplex formation with potential implications for branch point adenosine recognition fidelity.

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A Stochastic View of Spliceosome Assembly and Recycling in the Nucleus

Cited by 58 publications

References 58 publications

Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis

Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis

The timing of pre-mRNA splicing visualized in real-time

Structural and mechanistic insights into human splicing factor SF3b complex derived using an integrated approach guided by the cryo-EM density maps

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