Characterization of the roles of yeast nuclear exosome cofactor TRAMP complex in pre-mRNA splicing

2013 ◽  
Author(s):  
Ka-yiu Kong
Keyword(s):  
Mrna Splicing ◽  
Tramp Complex ◽  
Nuclear Exosome

Detection and characterization of a factor which rescues spliceosome assembly from a heat-inactivated HeLa cell nuclear extract

1991 ◽  
Vol 11 (7) ◽  
pp. 3425-3431
Author(s):  
P Delannoy ◽  
M H Caruthers
Keyword(s):  
Heat Treatment ◽  
Hela Cell ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Nuclear Extracts ◽  
Small Nuclear Rnas ◽  
A Minor ◽  
Cell Nuclear Extract

Mild heat treatment of HeLa cell nuclear extracts (NE) selectively inhibits pre-mRNA splicing. Heat-inactivated extracts can be complemented by a small amount of untreated NE. Utilizing this complementation assay and a combination of ion-exchange, affinity, and hydrophobic chromatography, a heat reversal factor (HRF) was purified from NE that is required to rescue pre-mRNA splicing from a heat-inactivated extract. This activity in its most purified form consistently copurified in a fraction containing two 70-kDa proteins and a minor polypeptide of approximately 100 kDa. It was free of the major small nuclear RNAs, sensitive to protease, and required to rescue spliceosome formation from a heat-inactivated nuclear extract. These results suggest that this factor is a protein that may be an important component in pre-mRNA splicing, or alternatively, it may be involved in renaturation of a heat-sensitive splicing factor.

scholarly journals Characterization of two novel intronic OPA1 mutations resulting in aberrant pre-mRNA splicing

2017 ◽  
Vol 18 (1) ◽  
Author(s):  
Ramona Bolognini ◽  
Christina Gerth-Kahlert ◽  
Mathias Abegg ◽  
Deborah Bartholdi ◽  
Nicolas Mathis ◽  
...  
Keyword(s):  
Mrna Splicing

scholarly journals A non-invasive diagnostic assay for rapid detection and characterization of aberrant mRNA-splicing by nonsense mediated decay inhibition

2020 ◽  
Vol 130 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Friederike Häuser ◽  
Seyfullah Gökce ◽  
Gesa Werner ◽  
Sven Danckwardt ◽  
Stefanie Sollfrank ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Mrna Splicing ◽  
Diagnostic Assay ◽  
Nonsense Mediated Decay ◽  
Non Invasive

scholarly journals A Yeast Exosome Cofactor, Mpp6, Functions in RNA Surveillance and in the Degradation of Noncoding RNA Transcripts

2008 ◽  
Vol 28 (17) ◽  
pp. 5446-5457 ◽  
Author(s):  
Laura Milligan ◽  
Laurence Decourty ◽  
Cosmin Saveanu ◽  
Juri Rappsilber ◽  
Hugo Ceulemans ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Rna Binding ◽  
Rna Surveillance ◽  
Synthetic Lethal ◽  
A Genome ◽  
Intergenic Regions ◽  
High Conservation ◽  
Tramp Complex ◽  
Nuclear Exosome ◽  
Functional Analyses

ABSTRACT A genome-wide screen for synthetic lethal (SL) interactions with loss of the nuclear exosome cofactors Rrp47/Lrp1 or Air1 identified 3′→5′ exonucleases, the THO complex required for mRNP assembly, and Ynr024w (Mpp6). SL interactions with mpp6Δ were confirmed for rrp47Δ and nuclear exosome component Rrp6. The results of bioinformatic analyses revealed homology between Mpp6 and a human exosome cofactor, underlining the high conservation of the RNA surveillance system. Mpp6 is an RNA binding protein that physically associates with the exosome and was localized throughout the nucleus. The results of functional analyses demonstrated roles for Mpp6 in the surveillance of both pre-rRNA and pre-mRNAs and in the degradation of “cryptic” noncoding RNAs (ncRNAs) derived from intergenic regions and the ribosomal DNA spacer heterochromatin. Strikingly, these ncRNAs are also targeted by other exosome cofactors, including Rrp47, the TRAMP complex (which includes Air1), and the Nrd1/Nab3 complex, and are degraded by both Rrp6 and the core exosome. Heterochromatic transcripts and other ncRNAs are characterized by very rapid degradation, and we predict that functional redundancy is an important feature of ncRNA metabolism.

scholarly journals Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency

2017 ◽  
Vol 114 (18) ◽  
pp. 4739-4744 ◽  
Author(s):  
Megan Mayerle ◽  
Madhura Raghavan ◽  
Sarah Ledoux ◽  
Argenta Price ◽  
Nicholas Stepankiw ◽  
...  
Keyword(s):  
High Efficiency ◽  
Catalytic Efficiency ◽  
Mrna Splicing ◽  
Catalytic Core ◽  
Eukaryotic Gene ◽  
Functional Classes ◽  
Reporter Assays

Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the “master regulator” of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome’s catalytic core and demonstrate that prp8 alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, the biological relevance of which are unknown. We performed an extensive characterization of prp8 alleles that map to this extension and, using in vitro and in vivo reporter assays, show they fall into two functional classes associated with the two structures: those that promote error-prone/efficient splicing and those that promote hyperaccurate/inefficient splicing. Identification of global locations of endogenous splice-site activation by lariat sequencing confirms the fidelity effects seen in our reporter assays. Furthermore, we show that error-prone/efficient RH alleles suppress a prp2 mutant deficient at promoting the first catalytic step of splicing, whereas hyperaccurate/inefficient RH alleles exhibit synthetic sickness. Together our data indicate that prp8 RH alleles link splicing fidelity with catalytic efficiency by biasing the relative stabilities of distinct spliceosome conformations. We hypothesize that the spliceosome “toggles” between such error-prone/efficient and hyperaccurate/inefficient conformations during the splicing cycle to regulate splicing fidelity.

scholarly journals Myb-Related Fission Yeast cdc5p Is a Component of a 40S snRNP-Containing Complex and Is Essential for Pre-mRNA Splicing

1999 ◽  
Vol 19 (8) ◽  
pp. 5352-5362 ◽  
Author(s):  
W. Hayes McDonald ◽  
Ryoma Ohi ◽  
Natalia Smelkova ◽  
David Frendewey ◽  
Kathleen L. Gould
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Fission Yeast ◽  
Genetic Interaction ◽  
Cell Cycle Control ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Immunoaffinity Purification ◽  
Snrnp Protein

ABSTRACT Myb-related cdc5p is required for G2/M progression in the yeast Schizosaccharomyces pombe. We report here that all detectable cdc5p is stably associated with a multiprotein 40S complex. Immunoaffinity purification has allowed the identification of 10 cwf (complexed with cdc5p) proteins. Two (cwf6p and cwf10p) are members of the U5 snRNP; one (cwf9p) is a core snRNP protein. cwf8p is the apparent ortholog of the Saccharomyces cerevisiaesplicing factor Prp19p. cwf1 + is allelic to theprp5 + gene defined by the S. pombesplicing mutant, prp5-1, and there is a strong negative genetic interaction between cdc5-120 andprp5-1. Five cwfs have not been recognized previously as important for either pre-mRNA splicing or cell cycle control. Further characterization of cwf1p, cwf2p, cwf3p, and cwf4p demonstrates that they are encoded by essential genes, cosediment with cdc5p at 40S, and coimmunoprecipitate with cdc5p. We further show that cdc5p associates with the U2, U5, and U6 snRNAs and that cells lackingcdc5 + function are defective in pre-mRNA splicing. These data raise the possibility that the cdc5p complex is an intermediate in the assembly or disassembly of an active S. pombe spliceosome.

scholarly journals ADSPPMutation Causing Dentinogenesis Imperfecta and Characterization of the Mutational Effect

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Sook-Kyung Lee ◽  
Kyung-Eun Lee ◽  
Su Jeong Song ◽  
Hong-Keun Hyun ◽  
Sang-Hoon Lee ◽  
...  
Keyword(s):  
Candidate Gene ◽  
Protein Secretion ◽  
Mutational Analysis ◽  
Gene Sequencing ◽  
Mrna Splicing ◽  
Dentinogenesis Imperfecta ◽  
Exon 2 ◽  
Dentin Matrix ◽  
Candidate Gene Sequencing

Mutations in theDSPPgene have been identified in nonsyndromic hereditary dentin defects, but the genotype-phenotype correlations are not fully understood. Recently, it has been demonstrated that the mutations ofDSPPaffecting the IPV leader sequence result in mutant DSPP retention in rough endoplasmic reticulum (ER). In this study, we identified a Korean family with dentinogenesis imperfecta type III. To identify the disease causing mutation in this family, we performed mutational analysis based on candidate gene sequencing. Exons and exon-intron boundaries ofDSPPgene were sequenced, and the effects of the identified mutation on the pre-mRNA splicing and protein secretion were investigated. Candidate gene sequencing revealed a mutation (c.50C > T, p.P17L) in exon 2 of theDSPPgene. The splicing assay showed that the mutation did not influence pre-mRNA splicing. However, the mutation interfered with protein secretion and resulted in the mutant protein remaining largely in the ER. These results suggest that the mutation affects ER-to-Golgi apparatus export and results in the reduction of secreted DSPP and ER overload. This may induce cell stress and damage processing and/or transport of dentin matrix proteins or other critical proteins.

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