The exonuclease ERI-1 has a conserved dual role in 5.8S rRNA processing and RNAi

Abstract

The exonuclease ERI-1 negatively regulates RNA interference (RNAi) in Caenorhabiditis elegans and Schizosaccharomyces pombe, and is required for production of some C. elegans endogenous small-interfering RNAs. We show that ERI-1 performs 3′ end processing of the 5.8S ribosomal RNA (rRNA) in both C. elegans and S. pombe. In C. elegans, two protein isoforms of ERI-1 are localized to the cytoplasm, and each has distinct functions in rRNA processing and negative regulation of RNAi.The C. elegans eri-1 gene encodes a 3′ to 5′ exonuclease of the DEDDh superfamily of RNase T exonucleases that was identified as a negative regulator of RNA interference (RNAi) 1 . Mutations in eri-1 cause an enhanced RNA interference (Eri) phenotype by which double stranded RNAs (dsRNAs) that are ineffective in silencing target mRNAs in wildtype animals trigger robust silencing in the Eri mutant. In the fission yeast S. pombe loss of Eri1 causes increased levels of small interfering RNAs (siRNAs) corresponding to centromeric repeats and a concomitant increase in RNAi-dependent heterochromatin formation at these genomic loci 2 . Analysis of ERI-1 in C. elegans, human and fission yeast has shown that it can degrade the 3′ end of siRNAs and histone mRNAs in vitro [1][2][3][4] , but in vivo substrates for this conserved enzyme are poorly understood.In the course of the analysis of RNAs isolated from the eri-1 null mutant, we observed that the 5.8S rRNA in an eri-1 worm is longer than wild-type 5.8S rRNA (Fig. 1a). This length difference is present in all detectable 5.8S rRNA, suggesting that eri-1 mutants have an rRNA processing defect in most, if not all, cells. The mature 5.8S, 18S, and 25-28S rRNAs in eukaryotes are generated from a 35S-47S precursor RNA via a series of processing steps mediated by multiple nucleases 5 . The activity of ERI-1 as a 3′ to 5′ exonuclease [1][2][3][4] suggests that the longer 5.8S rRNA is due to an extension of the 3′ end. RNase H cleavage and 3′ end cloning on the 5.8S rRNA of wild-type and mutant C. elegans indicated that all eri-1 5.8S rRNA is at least 1 nucleotide longer than the wild-type 5.8S rRNA specifically at the 3′ end, with a substantial fraction of eri-1 5.8S rRNA containing 2 to 4 additional nucleotides ( Supplementary Fig. 1 online). The 5.8S processing defect was observed in two independently derived eri-1 alleles, including another null allele (mg388, data not shown), and is rescued by an eri-1 transgene (see below), proving that loss of eri-1 causes the 5.8S processing defect. The 5.8S processing defect was not observed in other enhanced RNAi mutants with pleiotropies in common with eri-11 ,6 (data not shown). To characterize whether ERI-1 function in rRNA trimming is an ancient feature of this orthology group, we examined the 5.8S rRNA in the S. pombe erilΔ mutant and observed a length defect similar to the C. elegans eri-1 mutant (Fig. 1b). RNase H and sequencing analysis revealed a 3′ extended 5.8S rRNA species in erilΔ containing from 2 to 8 additional 3′ nucleotides ( Supplement...

Mouse Eri1 interacts with the ribosome and catalyzes 5.8S rRNA processing

“…Eri1 associates with ribosomes and binds to rRNA precursors and 5.8S rRNA in cells. The appearance of a 3′-extended 5.8S rRNA in C. elegans eri-1 mutants indicates that ERI-1 is required for 5.8S rRNA processing 10 , and our observation of a similar defect in Eri1-deficient mice demonstrates that this is a conserved feature of Eri1 function. Reconstitution experiments in Eri1-deficient cells and in vitro processing reactions with recombinant Eri1 revealed that Eri1 exonuclease activity is necessary and sufficient to catalyze the final step in 5.8S rRNA 3′ end formation.…”

“…Yet our understanding of ribosome biogenesis remains incomplete. In the accompanying manuscript, Gabel and Ruvkun demonstrate that eri-1 is essential for the maturation of the 3′ end of 5.8S rRNA in fission yeast and nematodes 10 . Together with our findings in mice, these results establish Eri1 as a conserved component of the rRNA processing machinery from fission yeast to mice.…”

“…On the basis of the finding that the 5.8S rRNA of C. elegans eri-1 mutants migrates more slowly in polyacrylamide gels 10 , we looked closely and observed a similar defect in Eri1 −/− mouse tissues and cultured cells (Fig. 3a, Supplementary Fig.…”

“…However, functional Eri1-GFP fusion proteins seem to be strictly cytoplasmic in C. elegans, and Flag-tagged Eri1 localized only to the cytoplasm in S. pombe 1,4,10 . Therefore, Eri1-mediated 5.8S rRNA maturation may occur after ribosome assembly and export to the cytoplasm, at least in these species.…”

“…Because Eri1 encodes a DEDDh-type exonuclease with RNase activity toward the 3′ ends of RNA in vitro 1,[4][5][6] , it was proposed that Eri1 counteracts the RNAi pathway by degrading siRNAs 7 . Alternatively, Eri1 may inhibit RNAi indirectly by sequestering factors involved in siRNA processing into a different protein complex [8][9][10] .…”