mRNA splicing efficiency in yeast and the contribution of nonconserved sequences

Pikielny, Claudio W.; Rosbash, Michael

doi:10.1016/0092-8674(85)90066-2

Cited by 180 publications

(147 citation statements)

References 22 publications

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“…We found that ctk1Δ mutants are indeed defective in the splicing of the newly synthesized RPS17A transcript, which is thought to be cotranscriptionally spliced very efficiently (82,83). In fact, under our assay conditions, we have never observed unspliced RPS17A pre-mRNA in wild-type cells.…”

Section: Pcaps and Their Phosphoctd-interacting Domains (Pc-ids): Funmentioning

confidence: 56%

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

2004

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CTD kinase I (CTDK-I) of Saccharomyces cerevisiae is required for normal phosphorylation of the C-terminal repeat domain (CTD) on elongating RNA polymerase II. To elucidate cellular roles played by this kinase and the hyperphosphorylated CTD (phosphoCTD) it generates, we systematically searched yeast extracts for proteins that bound to the phosphoCTD made by CTDK-I in vitro. Initially, using a combination of far-western blotting and phosphoCTD affinity chromatography, we discovered a set of novel phosphoCTD-associating proteins (PCAPs) implicated in a variety of nuclear functions. We identified the phosphoCTD-interacting domains of a number of these PCAPs, and in several test cases (namely, Set2, Ssd1, and Hrr25) adduced evidence that phosphoCTD binding is functionally important in vivo. Employing surface plasmon resonance (BIACORE) analysis, we found that recombinant versions of these and other PCAPs bind preferentially to CTD repeat peptides carrying SerPO 4 residues at positions 2 and 5 of each seven amino acid repeat, consistent with the positional specificity of CTDK-I in vitro [Jones, J. C., et al. (2004) J. Biol. Chem. 279, 24957-24964]. Subsequently, we used a synthetic CTD peptide with three doubly phosphorylated repeats (2,5P) as an affinity matrix, greatly expanding our search for PCAPs. This resulted in identification of approximately 100 PCAPs and associated proteins representing a wide range of functions (e.g., transcription, RNA processing, chromatin structure, DNA metabolism, protein synthesis and turnover, RNA degradation, snRNA modification, and snoRNP biogenesis). The varied nature of these PCAPs and associated proteins points to an unexpectedly diverse set of connections between Pol II elongation and other processes, conceptually expanding the role played by CTD phosphorylation in functional organization of the nucleus.The carboxyl-terminal repeat domain (CTD) 1 of the largest subunit of eukaryotic RNA polymerase II (Pol II) comprises tandem repeats of the consensus heptapeptide YSPTSPS. This highly conserved sequence, which is repeated 26 times in yeast and 52 times in mammals, is essential for viability. Phosphorylation of the CTD regulates the activities of the polymerase: only Pol II with an unphosphorylated CTD (Pol IIA) is thought to be able to assemble into preinitiation complexes at the promoter, whereas elongating polymerase has a hyperphosphorylated CTD (Pol IIO) (1,2). The serines at positions 2 and 5 within the heptad repeat represent major sites of phosphorylation during transcription. † Supported by National Institutes of Health Grant GM40505.© 2004 American Chemical Society * To whom correspondence should be addressed. . E-mail: arno@biochem.duke.edu. 1 Abbreviations: CTDK-I, CTD kinase I; Pol II, RNA polymerase II; CTD, C-terminal repeat domain; phosphoCTD, hyperphosphorylated CTD; PCTD, phosphoCTD; PCAP, phosphoCTD-associating protein; PCID, phosphoCTD-interacting domain; SGD, Saccharomyces Genome Database; SPR, surface plasmon resonance; RU, response units. NIH Publi...

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Section: Pcaps and Their Phosphoctd-interacting Domains (Pc-ids): Funmentioning

confidence: 56%

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

2004

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“…A 23-residue oligonucleotide complementary to the 5' end of exon 2 was used as primer. The 5'-end-labeled primer was hybridized to RNA overnight and the reverse transcription was performed as described (31) except that 0.2 mM ddATP or ddGTP was used and the three other dNTPs were present at 0.08 mM (32).…”

Section: Methodsmentioning

confidence: 99%

“…Primer extension analysis ofthe total processed RNA from each transcript was performed in the presence of a dideoxynucleoside triphosphate (ddXTP) and the three other dNTPs (32). A 23-residue oligonucleotide complementary to the 5' end of exon 2 was used as primer.…”

Section: Methodsmentioning

confidence: 99%

A 5' splice-region G----C mutation in exon 1 of the human beta-globin gene inhibits pre-mRNA splicing: a mechanism for beta+-thalassemia.

Vidaud

Gattoni

Stévenin

et al. 1989

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

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We have characterized a Mediterranean ,Bthalassemia allele containing a sequence change at codon 30 that alters both ,B-globin pre-mRNA splicing and the structure of the hemoglobin product. Presumably, this G -. C transversion at position -1 of intron 1 reduces severely the utilization of the normal 5' splice site since the level of the Arg -. Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position -1 of the CAG/GTAAGT consensus sequence had been isolated previously, we investigated the role of this nucleotide in the constitution of an active 5' splice site by studying the splicing of the pre-mRNA in cell-free extracts. We demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Our results provide further insights into the mechanisms ofpre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5. and 3).The splicing of mRNA precursors requires at least two conserved sequences at the ends of introns: the 5' splice-site consensus sequence CAG/GTAAGT, which shows a striking complementarity to the 5'-terminal region ofU1 small nuclear RNA (snRNA), and the 3' splice-site consensus sequence Y"NCAG/ (4-6). Most of the naturally occurring mutations and in vitro-generated mutations that reduce the complementarity with U1 snRNA result in authentic 5' splice-site inactivation (for reviews, see refs. 7 and 8) and can activate cryptic sites (9-11). Biochemical data and complementation experiments also support the proposal that the 5' end of U1 snRNA interacts with the 5' splice site in mRNA precursors (12)(13)(14)(15). However, it is not possible to predict with confidence the effect of one mutation at a given 5' splice site. For instance, a number of point mutations in the 5' splice site of the large rabbit 3-globin intron do not severely inhibit splicing in transfected Hela cells or in vitro (10,16,17). These results could be interpreted by proposing that 5' splice sites whose sequences match best with the consensus can undergo point mutation without severe inactivation (18,19). Nevertheless, the strength of the 5' splice sites is probably not the only factor involved in the splice-site selection, which can be less dependent on match to the consensus than on the sequence context in which the 5' splice site resides (20). A possible explanation for these divergent observations could be that the individual contribution of exon or intron residues to the 5' splice site is not equivalent. However, the role of the nucleotides preceding the 5' splice site, which are moderately conserved, has not been extensively documented (8). It is striking that no natural mutations of the guanine at position -1 (present in 80% of cases for human genes) have been isolated.Here we describe the isolation and characterization of a G C mutation in the consensus sequence at the last nucleotide of exon 1...

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“…Immunoprecipitation experiments with anti-PRP4 antibodies were performed as described (22) (45). RNAs were separated on a 5% polyacrylamide:bis-acrylamide (19:1), 7 M urea gel in 1 xTBE and electroblotted (semi-dry electro-blotting) onto nylon membrane (Hybond N, Amersham or Positive'm, Appligene) at 2.5 mA per cm2 for 20 min.…”

Section: Immunoprecipitation Experimentsmentioning

confidence: 99%

The biochemical defects ofprp4-1andprp6-1yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP

Galisson

Legrain

1993

Nucl Acids Res

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mRNA splicing efficiency in yeast and the contribution of nonconserved sequences

Cited by 180 publications

References 22 publications

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

A 5' splice-region G----C mutation in exon 1 of the human beta-globin gene inhibits pre-mRNA splicing: a mechanism for beta+-thalassemia.

The biochemical defects ofprp4-1andprp6-1yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP

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