Saccharomyces cerevisiae RAI1 (YGL246c) Is Homologous to Human DOM3Z and Encodes a Protein That Binds the Nuclear Exoribonuclease Rat1p

Abstract

The RAT1 gene of Saccharomyces cerevisiae encodes a 533 exoribonuclease which plays an essential role in yeast RNA degradation and/or processing in the nucleus. We have cloned a previously uncharacterized gene (YGL246c) that we refer to as RAI1 (Rat1p interacting protein 1). RAI1 is homologous to Caenorhabditis elegans DOM-3 and human DOM3Z. Deletion of RAI1 confers a growth defect which can be complemented by an additional copy of RAT1 on a centromeric vector or by directing Xrn1p, the cytoplasmic homolog of Rat1p, to the nucleus through the addition of a nuclear targeting sequence. Deletion of RAI1 is synthetically lethal with the rat1-1 ts mutation and shows genetic interaction with a deletion of SKI2 but not XRN1. Polysome analysis of an rai1 deletion mutant indicated a defect in 60S biogenesis which was nearly fully reversed by high-copy RAT1. Northern blot analysis of rRNAs revealed that rai1 is required for normal 5.8S processing. In the absence of RAI1, 5.8S L was the predominant form of 5.8S and there was an accumulation of 3-extended forms but not 5-extended species of 5.8S. In addition, a 27S pre-rRNA species accumulated in the rai1 mutant. Thus, deletion of RAI1 affects both 5 and 3 processing reactions of 5.8S rRNA. Consistent with the in vivo data suggesting that RAI1 enhances RAT1 function, purified Rai1p stabilized the in vitro exoribonuclease activity of Rat1p.In the yeast Saccharomyces cerevisiae, two 5Ј33Ј exoribonucleases, Rat1p and Xrn1p (23,35), are functionally interchangeable and are localized to the nucleus and cytoplasm, respectively (19). These enzymes are conserved throughout eukaryotes but are apparently absent from prokaryotic cells based on analysis of protein sequences predicted from prokaryotic genomes. The mammalian counterparts of Rat1p and Xrn1p function in yeast and complement their respective mutants (8,33,34). These exoribonucleases processively degrade RNA containing a 5Ј-monophosphate (37, 38). RNA molecules with 7mG cap, 5Ј-triphosphate, or 5Ј-hydroxyl are resistant to degradation by these enzymes (38). The role of Xrn1p in general deadenylation-dependent mRNA degradation and in nonsense-mediated degradation is well established (18,26,27). Xrn1p also degrades the 5Ј fragment of internal transcribed spacer 1, resulting from cleavage at sites D and A 2 (36; reviewed in reference 40). Xrn1p is not an essential protein, probably reflecting its partial redundancy with factors required for a 3Ј degradation pathway (6, 21).On the other hand, Rat1p activity in the nucleus is essential. The enzyme acts in numerous RNA 5Ј processing reactions important for ribosome biogenesis. These include maturation of 5.8S rRNA (16) and 5Ј trimming of intron-encoded and polycistronic small nucleolar RNAs (snoRNAs) (30, 32). Interestingly, many of the known Rat1p-mediated reactions can be accomplished to some extent by Xrn1p, and thus, the observation of processing intermediates requires double mutants of XRN1 and RAT1 (30,32).RAT1 has been identified from a variety of genetic and biochemical ap...

Torpedo Nuclease Rat1 Is Insufficient to Terminate RNA Polymerase II in Vitro

“…This work provided Rat1 and the complexes Rat1-Rai1 and Rat1-Rai1-Rtt103 in a pure, bacterially expressed, and recombinant form. The results also confirmed the protein-protein interactions that were previously inferred from co-purifications of endogenous proteins (9, 16) and coexpression of Rat1 and Rai1 in yeast (10,38).…”

“…pol II shows termination defects in yeast cells that are deficient in Rat1 or lack Rai1 (9), a factor that binds Rat1 in vivo and stabilizes the Rat1 exonuclease activity in vitro (10). Similar termination defects occur in mammalian cells after knockdown of the Rat1 homolog Xrn2 (11,12).…”

Transcription termination by nuclear RNA polymerases

“…Most commonly, chromatin remodeling occurs via histone modifications (Berger 2002) or ATPdependant DNA remodeling complexes (Narlikar et al 2002). Chromatin remodeling factors, grouped into three different families (SWI/SNF, ISWI, and CHD/Mi2), play crucial roles in transcription initiation (Xue et al 2000). However, evidence has accumulated that these factors may also function in transcription termination.…”

mRNA stability in the nucleus

“…Rat1p forms a stable complex with Rai1p that stimulates its exoribonuclease activity (Stevens and Poole, 1995;Xue et al, 2000;Xiang et al, 2009). The complex associates with a C-terminal domain binding protein, Rrt103p, and then contributes to RNP II termination in yeast.…”