Rna14p, a component of the yeast nuclear cleavage/polyadenylation factor I, is also localised in mitochondria

RNA14 was identified as a gene involved in premessenger RNA cleavage and polyadenylation. These processing steps take place in the nucleus, but the Rna14p protein is distributed in both the nucleus and the cytoplasm. By subcellular fractionation, we show here that the cytoplasmic fraction is localised in the mitochondria. In order to understand the role played by Rna14p in mitochondria, we have searched for new thermosensitive alleles of RNA14. We isolated thirteen new mutants. Some of them are deficient in mRNA cleavage and polyadenylation at the restrictive temperature - like the first mutant identified (rna14-1). However, others do not appear to be impaired in any of the steps in RNA metabolism investigated, nor do they appear to be involved in the replication or expression of mitochondrial DNA or in respiration. The localisation data strongly suggest that, besides an essential function in mRNA polyadenylation, the Rna14p protein has a non essential function in mitochondrial metabolism.     

Rna14p, a component of the yeast nuclear Cleavage / Polyadenylation Factor I, is also localised in mitochondria

RNA14 was identified as a gene involved in premessenger RNA cleavage and polyadenylation. These processing steps take place in the nucleus, but the Rna14p protein is distributed in both the nucleus and the cytoplasm. By subcellular fractionation, we show here that the cytoplasmic fraction is localised in the mitochondria. In order to understand the role played by Rna14p in mitochondria, we have searched for new thermosensitive alleles of RNA14. We isolated thirteen new mutants. Some of them are deficient in mRNA cleavage and polyadenylation at the restrictive temperature – like the first mutant identified (rna14-1). However, others do not appear to be impaired in any of the steps in RNA metabolism investigated, nor do they appear to be involved in the replication or expression of mitochondrial DNA or in respiration. The localisation data strongly suggest that, besides an essential function in mRNA polyadenylation, the Rna14p protein has a non essential function in mitochondrial metabolism.     

Yeast Pab1 interacts with Rna15 and participates in the control of the poly(A) tail length in vitro.

In Saccharomyces cerevisiae, the single poly(A) binding protein, Pab1, is the major ribonucleoprotein associated with the poly(A) tails of mRNAs in both the nucleus and the cytoplasm. We found that Pab1 interacts with Rna15 in two-hybrid assays and in coimmunoprecipitation experiments. Overexpression of PAB1 partially but specifically suppressed the rna15-2 mutation in vivo. RNA15 codes for a component of the cleavage and polyadenylation factor CF I, one of the four factors needed for pre-mRNA 3'-end processing. We show that Pab1 and CF I copurify in anion-exchange chromatography. These data suggest that Pab1 is physically associated with CF I. Extracts from a thermosensitive pab1 mutant and from a wild-type strain immunoneutralized for Pab1 showed normal cleavage activity but a large increase in poly(A) tail length. A normal tail length was restored by adding recombinant Pab1 to the mutant extract. The longer poly(A) tails were not due to an inhibition of exonuclease activities. Pab1 has previously been implicated in the regulation of translation initiation and in cytoplasmic mRNA stability. Our data indicate that Pab1 is also a part of the 3'-end RNA-processing complex and thus participates in the control of the poly(A) tail lengths during the polyadenylation reaction.     

Mutations inSTS1 suppress the defect in 3′ mRNA processing caused by therna15-2 mutation inSaccharomyces cerevisiae

In a search for proteins associated with Rna15p in processing the 3′ ends of messenger RNAs, we have looked for suppressors that correct, even partially, the thermosensitive growth defect of therna15-2 mutant. Mutations in a single locus that we namedSSM5, were able to suppress both the thermosensitivity of cell growth and the mRNA 3′ processing defect associated with therna15-2 mutation, but only slightly alleviated the thermosensitive growth defect of anrna14-1 mutant. Thessm5-1 mutant is sensitive to hydroxyurea at 37° C, a drug that inhibits DNA synthesis. By screening for complementation of the hydroxyurea-sensitive phenotype we cloned the corresponding wild-type gene and found that it corresponds to the essential geneSTS1 (also namedDBF8). Sts1p has an apparent molecular weight of 30 kDa and was confirmed to be a cytosolic protein by immunofluorescence analysis. Western blot analysis indicates that the thermosensitive mutant strainsrna15-2, rna14-1 andpap1-1 present a very low level of the Rna15p at 37° C. Thessm5-1 mutation restores the level of Rna15p in therna15-2 ssm5-1 double mutant. Use of the two-hybrid system suggests that Sts1p does not interact directly with Rna15p, but may be active as a homodimer. The present data suggest that Sts1p may play a role in the transport of Rna15p from the cytoplasm to the nucleus.     

Rna15 Interaction with the A-Rich Yeast Polyadenylation Signal Is an Essential Step in mRNA 3′-End Formation

In Saccharomyces cerevisiae, four factors [cleavage factor I (CF I), CF II, polyadenylation factor I (PF I), and poly(A) polymerase (PAP)] are required for maturation of the 3' end of the mRNA. CF I and CF II are required for cleavage; a complex of PAP and PF I, which includes CF II subunits, participates in polyadenylation, along with CF I. These factors are directed to the appropriate site on the mRNA by two sequences: one A-rich and one UA-rich. CF I contains five proteins, two of which, Rna15 and Hrp1, interact with the mRNA through RNA recognition motif-type RNA binding motifs. Previous work demonstrated that the UV cross-linking of purified Hrp1 to RNA required the UA-rich element, but the contact point of Rna15 was not known. We show here that Rna15 does not recognize a particular sequence in the absence of other proteins. However, in complex with Hrp1 and Rna14, Rna15 specifically interacts with the A-rich element. The Pcf11 and Clp1 subunits of CF I are not needed to position Rna15 at this site. This interaction is essential to the function of CF I. A mutant Rna15 with decreased affinity for RNA is defective for in vitro RNA processing and lethal in vivo, while an RNA with a mutation in the A-rich element is not processed in vitro and can no longer be UV cross-linked to the Rna15 subunit assembled into CF I. Thus, the recognition of the A-rich element depends on the tethering of Rna15 through an Rna14 bridge to Hrp1 bound to the UA-rich motif. These results illustrate that the yeast 3' end is defined and processed by a mechanism surprisingly different from that used by the mammalian system.     

Locked tether formation by cooperative folding of Rna14p monkeytail and Rna15p hinge domains in the yeast CF IA complex

The removal of the 3' region of pre-mRNA followed by polyadenylation is a key step in mRNA maturation. In the yeast Saccharomyces cerevisiae, one component of the processing machinery is the cleavage/polyadenylation factor IA (CF IA) complex, composed of four proteins (Clp1p, Pcf11p, Rna14p, Rna15p) that recognize RNA sequences adjacent to the cleavage site and recruit additional processing factors. To gain insight into the molecular architecture of CF IA we solved the solution structure of the heterodimer composed of the interacting regions between Rna14p and Rna15p. The C-terminal monkeytail domain from Rna14p and the hinge region from Rna15p display a coupled binding and folding mechanism, where both peptides are initially disordered. Mutants with destabilized monkeytail-hinge interactions prevent association of Rna15p within CF IA. Conservation of interdomain residues reveals that the structural tethering is preserved in the homologous mammalian cleavage stimulation factor (CstF)-77 and CstF-64 proteins of the CstF complex.     

Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import

The Saccharomyces cerevisiae gene, RNA1, encodes a protein with extensive homology to the mammalian Ran/TC4 GTPase activating protein. Using indirect immunofluorescence microscopy, we have demonstrated that rna1-1 mutant cells are defective in nuclear import of several proteins. The same result is obtained when nuclear import is examined in living cells using a nuclear protein fused to the naturally green fluorescent protein. These findings suggest a role for the Rna1p in trafficking of proteins across the nuclear membrane. To investigate this role more directly, an in vitro import assay that monitors the import of a fluorescently labeled substrate into the nuclei of semi- intact yeast cells was used. Import to the nucleus requires the addition of exogenous cytosol. Results indicate that, in contrast to wild-type cytosols, extracts made from rna1-1 mutant cells are unable to support import of the fluorescently labeled substrate into competent nuclei. Immunoblotting demonstrates that these mutant-derived extracts are depleted of Rna1p. However, when purified Rna1p is added back to these extracts the import activity is restored in a dose-dependent manner. These results demonstrate that Rna1p plays a direct role in the import of proteins into the nucleus.