Temperature-sensitive mutants of the exosome subunit Rrp43p show a deficiency in mRNA degradation and no longer interact with the exosome.

Rrp43p is a Saccharomyces cerevisiae exosome subunit involved in pre-rRNA processing which is found both in the nucleus and in the cytoplasm. So far, no function has been assigned to the cytoplasmic fraction of Rrp43p. We have addressed Rrp43p function by analyzing mRNA stability in three rrp43 temperature-sensitive (ts) strains, which carry different ts alleles (rrp43-1, rrp43-2 and rrp43-3), and by analyzing Rrp43p interactions with the remaining exosome subunits. In the ts strains, endogenous mRNAs (ACT1 and PAB1), as well as a heterologous reporter mRNA (CATpG) showed longer half-lives, relative to a control strain carrying wild-type RRP43. The mutants also accumulated a degradation intermediate of the reporter mRNA that is typical of defective mRNA decay. These results allow us to propose that Rrp43p is required for mRNA degradation. Rrp43p interacts with the exosome complex via Rrp46p, as determined by two-hybrid analyses. Interestingly, the rrp43 ts mutant proteins do not interact with Rrp46p, indicating that the ts phenotype may be caused by disruption of the Rrp43p- Rrp46p interaction. The ts strains also showed a pre-rRNA processing defect, which is consistent with previous studies on Rrp43p function.

[1]  R Parker,et al.  A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. , 1993, Genes & development.

[2]  P. Mitchell,et al.  Degradation of ribosomal RNA precursors by the exosome. , 2000, Nucleic acids research.

[3]  D. Tollervey,et al.  Identification of a Regulated Pathway for Nuclear Pre-mRNA Turnover , 2000, Cell.

[4]  F Sherman,et al.  Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis , 1997, Molecular and cellular biology.

[5]  Michael W. Briggs,et al.  Rrp6p, the Yeast Homologue of the Human PM-Scl 100-kDa Autoantigen, Is Essential for Efficient 5.8 S rRNA 3′ End Formation* , 1998, The Journal of Biological Chemistry.

[6]  F. Larimer,et al.  Fragments of the internal transcribed spacer 1 of pre-rRNA accumulate in Saccharomyces cerevisiae lacking 5'----3' exoribonuclease 1 , 1991, Journal of bacteriology.

[7]  J. Butler,et al.  A Nuclear 3′-5′ Exonuclease Involved in mRNA Degradation Interacts with Poly(A) Polymerase and the hnRNA Protein Npl3p , 2000, Molecular and Cellular Biology.

[8]  D. Goldfarb,et al.  The exosome subunit Rrp43p is required for the efficient maturation of 5.8S, 18S and 25S rRNA. , 1999, Nucleic acids research.

[9]  R. Parker,et al.  Three conserved members of the RNase D family have unique and overlapping functions in the processing of 5S, 5.8S, U4, U5, RNase MRP and RNase P RNAs in yeast , 2000, The EMBO journal.

[10]  K. Wu,et al.  Temperature sensitive nop2 alleles defective in synthesis of 25S rRNA and large ribosomal subunits in Saccharomyces cerevisiae. , 2001, Nucleic acids research.

[11]  C. Clayton,et al.  The exosome of Trypanosoma brucei , 2001, The EMBO journal.

[12]  G. Fink,et al.  Laboratory course manual for methods in yeast genetics , 1986 .

[13]  J. McCarthy,et al.  The Relationship between Eukaryotic Translation and mRNA Stability , 1995, The Journal of Biological Chemistry.

[14]  D. Tollervey,et al.  NOP3 is an essential yeast protein which is required for pre-rRNA processing , 1992, The Journal of cell biology.

[15]  R Parker,et al.  Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae , 1990, Molecular and cellular biology.

[16]  G. Pruijn,et al.  The mammalian exosome mediates the efficient degradation of mRNAs that contain AU‐rich elements , 2002, The EMBO journal.

[17]  E. Petfalski,et al.  The 5′ end of yeast 5.8S rRNA is generated by exonucleases from an upstream cleavage site. , 1994, The EMBO journal.

[18]  R. Sternglanz,et al.  Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system , 1995, Molecular and cellular biology.

[19]  P. Mitchell,et al.  Musing on the structural organization of the exosome complex , 2000, Nature Structural Biology.

[20]  P. Mitchell,et al.  mRNA stability in eukaryotes. , 2000, Current opinion in genetics & development.

[21]  M. Mann,et al.  The Exosome: A Conserved Eukaryotic RNA Processing Complex Containing Multiple 3′→5′ Exoribonucleases , 1997, Cell.

[22]  T. Geerlings,et al.  The final step in the formation of 25S rRNA in Saccharomyces cerevisiae is performed by 5'-->3' exonucleases. , 2000, RNA.

[23]  P. Mitchell,et al.  Functions of the exosome in rRNA, snoRNA and snRNA synthesis , 1999, The EMBO journal.

[24]  C L Hsu,et al.  Yeast cells lacking 5'-->3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure , 1993, Molecular and cellular biology.

[25]  R. Baker,et al.  Function of the Ski4p (Csl4p) and Ski7p Proteins in 3′-to-5′ Degradation of mRNA , 2000, Molecular and Cellular Biology.

[26]  E. Craig,et al.  Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. , 1996, Genetics.

[27]  T. Kuo,et al.  One-step transformation of yeast in stationary phase , 2004, Current Genetics.

[28]  P. Vreken,et al.  The rate-limiting step in yeast PGK1 mRNA degradation is an endonucleolytic cleavage in the 3'-terminal part of the coding region , 1992, Molecular and cellular biology.

[29]  M. Mann,et al.  AU Binding Proteins Recruit the Exosome to Degrade ARE-Containing mRNAs , 2001, Cell.

[30]  R Parker,et al.  Deadenylation of the unstable mRNA encoded by the yeast MFA2 gene leads to decapping followed by 5'-->3' digestion of the transcript. , 1994, Genes & development.

[31]  A. Vojtek,et al.  Ras-Raf interaction: two-hybrid analysis. , 1995, Methods in enzymology.

[32]  D. Tollervey,et al.  Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly , 1993, Cell.

[33]  G. Caponigro,et al.  Mechanisms and control of mRNA turnover in Saccharomyces cerevisiae. , 1996, Microbiological reviews.

[34]  Julia A Chekanova,et al.  Poly(A) Tail-dependent Exonuclease AtRrp41p fromArabidopsis thaliana Rescues 5.8 S rRNA Processing and mRNA Decay Defects of the Yeast ski6 Mutant and Is Found in an Exosome-sized Complex in Plant and Yeast Cells* , 2000, The Journal of Biological Chemistry.

[35]  D. Hartley.  Cellular interactions in development : a practical approach , 1993 .

[36]  J. Hegemann,et al.  Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast , 1996, Yeast.

[37]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[38]  J D Beggs,et al.  Characterization of Sm‐like proteins in yeast and their association with U6 snRNA , 1999, The EMBO journal.

[39]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[40]  S. Fields,et al.  The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[41]  E. Petfalski,et al.  Three Novel Components of the Human Exosome* , 2001, The Journal of Biological Chemistry.

[42]  M. Kiledjian,et al.  Functional Link between the Mammalian Exosome and mRNA Decapping , 2001, Cell.

[43]  D. Tollervey,et al.  Dob1p (Mtr4p) is a putative ATP‐dependent RNA helicase required for the 3′ end formation of 5.8S rRNA in Saccharomyces cerevisiae , 1998, The EMBO journal.

[44]  Roy Parker,et al.  The Exosome A Proteasome for RNA? , 1999, Cell.

[45]  R. Parker,et al.  Yeast Exosome Mutants Accumulate 3′-Extended Polyadenylated Forms of U4 Small Nuclear RNA and Small Nucleolar RNAs , 2000, Molecular and Cellular Biology.

[46]  R. Parker,et al.  A rapid method for localized mutagenesis of yeast genes , 1992, Yeast.

[47]  P. Mitchell,et al.  The 3' end of yeast 5.8S rRNA is generated by an exonuclease processing mechanism. , 1996, Genes & development.

[48]  R. Parker,et al.  The 3′ to 5′ degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3′ to 5′ exonucleases of the exosome complex , 1998, The EMBO journal.

[49]  J. Davies,et al.  Mode of Action of Thiolutin, an Inhibitor of Macromolecular Synthesis in Saccharomyces cerevisiae , 1973, Antimicrobial Agents and Chemotherapy.

[50]  D. Goldfarb,et al.  Nip7p Interacts with Nop8p, an Essential Nucleolar Protein Required for 60S Ribosome Biogenesis, and the Exosome Subunit Rrp43p , 1999, Molecular and Cellular Biology.

[51]  J. Warner,et al.  10 The Ribosome and Its Synthesis , 1991 .

[52]  A. Podtelejnikov,et al.  The yeast exosome and human PM-Scl are related complexes of 3' --> 5' exonucleases. , 1999, Genes & development.

[53]  D. Tollervey,et al.  Processing of pre‐ribosomal RNA in Saccharomyces cerevisiae , 1995, Yeast.