The Spliceosome: Design Principles of a Dynamic RNP Machine

Wahl, Markus C.; Will, Cindy L.; Lührmann, Reinhard

doi:10.1016/j.cell.2009.02.009

Cited by 2,304 publications

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“…Fourth, highly purified, fully assembled U2 or U4/U6•U5 tri-snRNP was capable of significant rescue of the CoilMO phenotype, whereas U1 snRNP was not. This suggests that stable U2 snRNP interactions with pre-mRNA, which trigger spliceosome assembly 6 , represent a rate-limiting step in embryonic gene expression. Because neither the snRNP proteins nor the snRNAs alone had rescuing ability and only mature assembled snRNPs were active, we conclude that macromolecular assembly of snRNPs is the essential function of coilin in zebrafish embryos.…”

Section: Discussionmentioning

confidence: 99%

“…Similarly to the tri-snRNP, intermediates in mature U2 snRNP formation can be trapped in CBs upon knockdown of appropriate assembly factors 17 . U2 snRNP is present in cells in 12S and 17S forms, reflecting the association of the core U2 snRNP containing the Sm ring U2A and U2B proteins (12S) with the SF3 complex (17S) 5,6 . Notably, both the 12S and 17S forms of the U2 snRNP rescued coilin morphants equally, indicating that formation of the 12S U2 snRNP is compromised upon coilin depletion.…”

Section: Discussionmentioning

confidence: 99%

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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%

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

152

164

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“…Recruitment of the U5 snRNP by the spliceosome is required for its catalytic activity. 1 RNA immunoprecipitation experiments using an anti-PRP6 antibody revealed that knockdown of SND1 correlated with a decreased recruitment of the U5 snRNP on the CD44 pre-mRNA in PC3 cells (Figure 5c), indicating that SND1 favors assembly of the active spliceosome. Moreover, SAM68 was also shown to associate with the U5 snRNP under conditions in which exon v5 inclusion was induced, 50 and we found that knockdown of SND1 strongly reduced the association of SAM68 with the CD44 pre-mRNA (Figure 5d).…”

Section: Identification Of Snd1 As a Novel Sam68-interacting Proteinmentioning

confidence: 99%

“…This phenomenon is driven by a large macromolecular complex, the spliceosome, composed of five small nuclear ribonucleoproteins (snRNPs) and over 200 auxiliary proteins. 1 In higher eukaryotes, a large number of exons can be alternatively spliced to yield different transcripts from a single gene, thereby increasing the coding potential of the genome. 2,3 Indeed, the large majority of multi-exon human genes undergo alternative splicing (AS) to produce at least two mRNA variants.…”

Section: Introductionmentioning

confidence: 99%

The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells

et al. 2013

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Splicing abnormalities have profound impact in human cancer. Several splicing factors, including SAM68, have pro-oncogenic functions, and their increased expression often correlates with human cancer development and progression. Herein, we have identified using mass spectrometry proteins that interact with endogenous SAM68 in prostate cancer (PCa) cells. Among other interesting proteins, we have characterized the interaction of SAM68 with SND1, a transcriptional co-activator that binds spliceosome components, thus coupling transcription and splicing. We found that both SAM68 and SND1 are upregulated in PCa cells with respect to benign prostate cells. Upregulation of SND1 exerts a synergic effect with SAM68 on exon v5 inclusion in the CD44 mRNA. The effect of SND1 on CD44 splicing required SAM68, as it was compromised after knockdown of this protein or mutation of the SAM68-binding sites in the CD44 pre-mRNA. More generally, we found that SND1 promotes the inclusion of CD44 variable exons by recruiting SAM68 and spliceosomal components on CD44 pre-mRNA. Inclusion of the variable exons in CD44 correlates with increased proliferation, motility and invasiveness of cancer cells. Strikingly, we found that knockdown of SND1, or SAM68, reduced proliferation and migration of PCa cells. Thus, our findings strongly suggest that SND1 is a novel regulator of alternative splicing that promotes PCa cell growth and survival.Oncogene advance online publication, 2 September 2013; doi:10.1038/onc.2013.360Keywords: alternative splicing; SND1; SAM68; CD44; prostate cancer INTRODUCTION Nuclear processing of pre-mRNAs requires tightly regulated steps that ultimately yield a mature and functional mRNA. Splicing is the step that insures the removal of long non-coding sequences (introns) from the pre-mRNA and the joining of the exons. This phenomenon is driven by a large macromolecular complex, the spliceosome, composed of five small nuclear ribonucleoproteins (snRNPs) and over 200 auxiliary proteins. 1 In higher eukaryotes, a large number of exons can be alternatively spliced to yield different transcripts from a single gene, thereby increasing the coding potential of the genome. 2,3 Indeed, the large majority of multi-exon human genes undergo alternative splicing (AS) to produce at least two mRNA variants. 4,5 As regulation of AS profoundly influences physiological and pathological processes, 3,6 the full comprehension of the molecular mechanisms regulating this step of pre-mRNA processing is of fundamental importance.Splicing is physically and functionally coupled to transcription. 7-10 Two models have been proposed for how transcription might affect changes in AS patterns. The 'recruitment model' suggests that the transcription apparatus physically interacts with splicing regulators, thereby affecting splicing decisions. The C-terminal domain (CTD) of the largest subunit of the RNA polymerase II (RNAPII) has a central role in this coupling process by favoring the recruitment of RNA processing factors on the nascent transcripts. 9,10 ...

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“…A large number of these molecular devices are based on nucleic acid scaff olds, for example, riboswitches 1 , ribozymes 2 , transposons 3 , ribosomes 4 , cis -regulatory elements on DNA 5 , spliceosomes 6 , to name a few. Owing to the specifi city and predictability of Watson-Crick base pairing, engineerability and the ability to form a rich repertoire of unusual structures based on their sequence and external triggers, nucleic acids have proven to be a remarkably powerful scaff old to build a variety of programmable synthetic nanomachines 7 .…”

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confidence: 99%

An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism

et al. 2011

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Structural DNA nanotechnology seeks to build synthetic molecular machinery from DNA. DNA nanomachines are artifi cially designed assemblies that switch between defi ned conformations in response to an external cue. Though it has proved possible to create DNA machines and rudimentary walkers, the function of such autonomous DNA-based molecular devices has not yet been achieved inside living organisms. Here we demonstrate the operation of a pH-triggered DNA nanomachine inside the nematode Caenorhabditis elegans . The nanomachine uses fl uorescence resonance energy transfer to effectively map spatiotemporal pH changes associated with endocytosis in wild type as well as mutant worms, demonstrating autonomous function within the organismal milieu in a variety of genetic backgrounds. From this fi rst demonstration of the independent functionality of a DNA nanomachine in vivo , we observe that rationally designed DNA-based molecular devices retain their in vitro functionality with quantitative precision. This positions DNA nanodevices as exciting and powerful tools to interrogate complex biological phenomena.

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The Spliceosome: Design Principles of a Dynamic RNP Machine

Abstract: Job Sharing and Cooperation between RNAs and Proteins RNPs are involved in a large spectrum of molecular activities. Whereas assemblies such as the signal recognition particle or the U7 snRNP serve as adaptors or transport devices, other

Cited by 2,304 publications

References 134 publications

Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis

Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis

The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells

An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism

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