Structure and Function of the Pre-mRNA Splicing Machine

Sperling, Joseph; Azubel, Maia; Sperling, Ruth

doi:10.1016/j.str.2008.08.011

Cited by 78 publications

(112 citation statements)

References 86 publications

(160 reference statements)

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“…In addition, multiple shorter fragments were present in the gradients, with apparent lengths of 22, 30, and 50 nt that reflected some of the fragments observed in the RNA-seq experiments. Some of these RNAs are present in fractions 8-11 that contain most of the in vivo spliceosomes (37,48) as determined by the presence of five spliceosomal U small nuclear ribonucleoproteins (snRNPs) (Fig. S1D) and regulatory splicing factors (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The gradient was subdivided into four parts: A-D. Fraction A contained proteins that sedimented faster than 200S and was devoid of spliceosomes; fraction B contained supraspliceosomes that sedimented in the 200S region (37,48,49), fraction C contained native spliceosomes that sedimented at 60-70S (37,41,50), and fraction D contained proteins and small RNAs that appeared as free proteins or assembled into small RNP complexes. SNORD2, SNORD60, and SNORD78 were present in fractions B and C associated with spliceosomes but devoid of fibrillarin (Figs.…”

Section: Resultsmentioning

confidence: 99%

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Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

Falaleeva

Pagès

Matuszek

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2′-Omethylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex, SNORD27 regulates the alternative splicing of the transcription factor E2F7 pre-mRNA through direct RNA-RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously "silent" exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes.alternative splicing | gene regulation | snoRNAs | pre-mRNA processing S mall nucleolar RNAs (snoRNAs) are 60-to 300-nt-long noncoding RNAs that accumulate in the nucleolus. Based on conserved sequence elements, snoRNAs are classified as C/D box small nucleolar RNAs (SNORDs) or H/ACA box snoRNAs (SNORAs). SNORDs contain sequence elements termed C (RUGAUGA) and D (CUGA) boxes, usually present in duplicates (C′ and D′ boxes), and up to two antisense boxes that hybridize to the target RNA (1). In humans, SNORDs are usually derived from introns. After the splicing reaction, introns are excised as lariats, which are then opened by the debranching enzyme and subsequently degraded. Intronic SNORDs escape this degradation by forming a protein complex that consists of non-histone chromosome protein 2-like 1 (NHP2L1, 15.5K, SNU13), nucleolar protein 5A (NOP56), nucleolar protein 5 (NOP58), and fibrillarin (2-4). The SNORD protein complex forms through the entry of the snoRNA and fibrillarin to a complex containing NHP2L1, NOP58, and at least five assembly factors (5). The SNORD acts as a scaffold for the final protein complex formation and also controls recognition of other RNAs using the antisense boxes. The antisense boxes recognize sequences in rRNA, resulting in the fifth nucleotide upstream of the D or D′ box being 2′-O-methylated by fibrillarin (1). Structural studies indicate that the active form of SNORDs is dimeric (6).The conserved overall structure of SNORDs allows the identification of their putative target RNA binding sites. However, numerous SNORDs without obvious target RNAs have been identified (7-10) and are termed "orphan snoRNAs." Genome-wide deep sequencing experiments identified shorter but stable SNORD fragments that were found in all species tested, ranging from mammals to the protozoan Giardia lamblia (11) and EpsteinBarr virus (12). Fragments longer than 27 nt generated by SNORDs will ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

Falaleeva

Pagès

Matuszek

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

171

152

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“…1 This process constitutes the removal of non-coding sequences (introns) from pre-mRNAs and subsequent ligation of the coding sequences (exons) to form a mature mRNA. [2][3][4] Several cis (pre-mRNA) and trans (snRNPs, accessory proteins) acting elements coordinate this process with high precision, to ensure fidelity. Higher eukaryotes contain a U2-dependent (major) splicing pathway and a less abundant U12-dependent (minor) pathway, which splices specific premRNA introns (U2-type and U12-type) differing in the splice site and branch point consensus sequences.…”

Section: Introductionmentioning

confidence: 99%

“…The U2-dependent pathway involves U1, U2, U5 and U4/U6 snRNPs while the U12-dependent pathway comprises of U11, U12, U5 and U4atac/U6atac snRNPs. 5 Multiple intermediary complexes (E, A, B, B act , B Ã and C) which are characterized by synergistic protein-protein, protein-RNA and RNA-RNA interactions 2,3 are involved in these pathways.…”

Section: Introductionmentioning

confidence: 99%

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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“…Sedimentation and co-fractionation experiments in, for example, maize have demonstrated that these proteins are part of large multiprotein and ribonucleoprotein complexes with their cognate RNAs (5,7). In addition, these complexes resemble the nuclear spliceosome in which the snRNAs associate with more than 200 proteins (8).…”

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confidence: 99%

Identification of a Chloroplast Ribonucleoprotein Complex Containing Trans-splicing Factors, Intron RNA, and Novel Components

Jacobs

Marx

Kock

et al. 2013

Molecular & Cellular Proteomics

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Maturation of chloroplast psaA pre-mRNA from the green alga Chlamydomonas reinhardtii requires the trans-splicing of two split group II introns. Several nuclear-encoded trans-splicing factors are required for the correct processing of psaA mRNA. Among these is the recently identified Raa4 protein, which is involved in splicing of the tripartite intron 1 of the psaA precursor mRNA. Part of this tripartite group II intron is the chloroplast encoded tscA RNA, which is specifically bound by Raa4. Using Raa4 as bait in a combined tandem affinity purification and mass spectrometry approach, we identified core components of a multisubunit ribonucleoprotein complex, including three previously identified trans-splicing factors (Raa1, Raa3, and Rat2). We further detected tscA RNA in the purified protein complex, which seems to be specific for splicing of the tripartite group II intron. Intron-containing genes from prokaryotic or organellar genomes carry either group I or group II introns, each of which has distinct features. The splicing mechanism of group II introns and the secondary structures of their presumed active sites were used as early arguments for the hypothesis that this class of introns represents the ancestors of eukaryotic spliceosomal introns (1, 2). It was further assumed that group II introns invaded the eukaryotic nucleus and subsequently proliferated at various genomic sites, leading to the degeneration of the catalytic intron structure into small nuclear RNAs (snRNAs).1 This assumption was supported by the observation of naturally occurring variants of group II introns that are split into two or more pieces (3), reminiscent of eukaryotic spliceosomal RNA (1). Group II intron RNAs are characterized by six conserved domains, and tertiary interactions among these domains generate the compact native and catalytic complex. Some of these group II intron domains have been shown to act in trans on the splicing of other introns that lack the corresponding domain (4). In vivo, various RNA-binding proteins promote the formation of catalytically active intron RNA. In contrast to the nuclear spliceosome, which acts generally on a broad range of nuclear-encoded pre-mRNAs, proteins involved in organellar intron splicing seem to more efficiently stabilize the active three-dimensional RNA structure in vivo. Several splicing factors in higher plants, such as the chloroplast RNA-splicing and ribosome maturation (CRM) domain protein CRS1, as well as the pentatricopeptide repeat proteins OTP51 and PPR4, have been reported to be involved in the splicing of single transcripts (5, 6). Nonetheless, there are splicing factors that carry out functions on a broad range of transcripts, including CRS2 and its associated proteins CAF1 and CAF2, and WTF1, a splicing factor containing a plant organelle RNA-recognition domain (5, 6). Sedimentation and co-fractionation experiments in, for example, maize have demonstrated that these proteins are part of large multiprotein and ribonucleoprotein complexes with their cognate RNAs (5, 7). I...

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Structure and Function of the Pre-mRNA Splicing Machine

Cited by 78 publications

References 86 publications

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

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

Identification of a Chloroplast Ribonucleoprotein Complex Containing Trans-splicing Factors, Intron RNA, and Novel Components

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