David Tollervey

The Exosome: A Conserved Eukaryotic RNA Processing Complex Containing Multiple 3′→5′ Exoribonucleases

We identified a complex in S. cerevisiae, the exosome, consisting of the five essential proteins Rrp4p, Rrp41p, Rrp42p, Rrp43p, and Rrp44p (Dis3p). Remarkably, four of these proteins are homologous to characterized bacterial 3-->5 exoribonucleases; Rrp44p is homologous to RNase II, while Rrp41p, Rrp42p, and Rrp43p are related to RNase PH. Recombinant Rrp4p, Rrp44p, and Rrp41p are 3-->5 exoribonucleases in vitro that have distributive, processive, and phosphorolytic activities, respectively. All components of the exosome are required for 3 processing of the 5.8S rRNA. Human Rrp4p is found in a comparably sized complex, and expression of the hRRP4 gene in yeast complements the rrp4-1 mutation. We conclude that the exosome constitutes a highly conserved eukaryotic RNA processing complex.

The 5' end of yeast 5.8S rRNA is generated by exonucleases from an upstream cleavage site.

We have developed techniques for the detailed analysis of cis‐acting sequences in the pre‐rRNA of Saccharomyces cerevisiae and used these to study the processing of internal transcribed spacer 1 (ITS1) leading to the synthesis of 5.8S rRNA. As is the case for many eukaryotes, the 5′ end of yeast 5.8S rRNA is heterogeneous; we designate the major, short form 5.8S(S), and the minor form (which is seven or eight nucleotides longer) 5.8S(L). These RNAs do not have a precursor/product relationship, but result from the use of alternative processing pathways. In the major pathway, a previously unidentified processing site in ITS1, designated A3, is cleaved. A 10 nucleotide deletion at site A3 strongly inhibits processing of A3 and the synthesis of 5.8S(S); processing is predominantly transferred to the alternative 5.8S(L) pathway. Site A3 lies 76 nucleotides 5′ to the end of 5.8S(S), and acts as an entry site for 5′‐‐>3′ exonuclease digestion which generates the 5′ end of 5.8S(S). This pathway is inhibited in strains mutant for XRN1p and RAT1p. Both of these proteins have been reported to have 5′‐‐>3′ exonuclease activity in vitro. Formation of 5.8S(L) is increased by mutations at A3 in cis or in RAT1p and XRN1p in trans, and is kinetically faster than 5.8S(S) synthesis.

Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes

Hal2p is an enzyme that converts pAp (adenosine 3′,5′ bisphosphate), a product of sulfate assimilation, into 5′ AMP and Pi. Overexpression of Hal2p confers lithium resistance in yeast, and its activity is inhibited by submillimolar amounts of Li+ in vitro. Here we report that pAp accumulation in HAL2 mutants inhibits the 5′→3′ exoribonucleases Xrn1p and Rat1p. Li+ treatment of a wild‐type yeast strain also inhibits the exonucleases, as a result of pAp accumulation due to inhibition of Hal2p; 5′ processing of the 5.8S rRNA and snoRNAs, degradation of pre‐rRNA spacer fragments and mRNA turnover are inhibited. Lithium also inhibits the activity of RNase MRP by a mechanism which is not mediated by pAp. A mutation in the RNase MRP RNA confers Li+ hypersensitivity and is synthetically lethal with mutations in either HAL2 or XRN1. We propose that Li+ toxicity in yeast is due to synthetic lethality evoked between Xrn1p and RNase MRP. Similar mechanisms may contribute to the effects of Li+ on development and in human neurobiology.

Yeast snR30 is a small nucleolar RNA required for 18S rRNA synthesis.

Subnuclear fractionation and coprecipitation by antibodies against the nucleolar protein NOP1 demonstrate that the essential Saccharomyces cerevisiae RNA snR30 is localized to the nucleolus. By using aminomethyl trimethyl-psoralen, snR30 can be cross-linked in vivo to 35S pre-rRNA. To determine whether snR30 has a role in rRNA processing, a conditional allele was constructed by replacing the authentic SNR30 promoter with the GAL10 promoter. Repression of snR30 synthesis results in a rapid depletion of snR30 and a progressive increase in cell doubling time. rRNA processing is disrupted during the depletion of snR30; mature 18S rRNA and its 20S precursor underaccumulate, and an aberrant 23S pre-rRNA intermediate can be detected. Initial results indicate that this 23S pre-rRNA is the same as the species detected on depletion of the small nucleolar RNA-associated proteins NOP1 and GAR1 and in an snr10 mutant strain. It was found that the 3 end of 23S pre-rRNA is located in the 3 region of ITS1 between cleavage sites A2 and B1 and not, as previously suggested, at the B1 site, snR30 is the fourth small nucleolar RNA shown to play a role in rRNA processing.

Birth of the snoRNPs: the evolution of RNase MRP and the eukaryotic pre-rRNA-processing system

The ribonucleoprotein particle RNase MRP is required for the processing of yeast pre-ribosomal RNA (pre-rRNA). A structurally related particle, RNase P, is universally required for processing of pre-tRNA, but in bacteria and archaea also cleaves a site in the pre-rRNA. This suggests that RNase MRP may have arisen in eukaryotes as a form of RNase P specialized for pre-rRNA processing. Other eukaryotic small nucleolar RNAs may have arisen as trans-acting factors that functionally replace cis-acting pre-rRNA interactions in bacteria and archaea.

Synthetic lethality with fibrillarin identifies NOP77p, a nucleolar protein required for pre-rRNA processing and modification.

The nucleolar protein fibrillarin (encoded by the NOP1 gene in yeast), is required for many post‐transcriptional steps in yeast ribosome synthesis. A screen for mutations showing synthetic lethality with a temperature sensitive nop1‐5 allele led to the identification of the NOP77 gene. NOP77 is essential for viability and encodes a nucleolar protein with a predicted molecular weight of 77 kDa. Depletion of NOP77p impairs both the processing and methylation of the pre‐rRNA. The processing defect is greatest for the pathway leading to 25S rRNA synthesis, and is distinctly different from that observed for mutations in other nucleolar components. NOP77p contains three canonical RNA recognition motifs (RRMs), suggesting that it is an RNA binding protein. The NOP77 allele which complements the synthetic lethal nop1 strains has an alanine at position 308, predicted to lie in helix alpha 1 of RRM3, whereas the non‐complementing nop77‐1 allele contains a proline at the corresponding position. We propose that NOP77p mediates specific interactions between NOP1p and the pre‐rRNA.

Small Nucleolar RNAs Guide Ribosomal RNA Methylation

Two recent reports, one in Cell and one in the Journal of Molecular Biology, show that the newly described small nucleolar RNAs control the site of methylation of the ribosomal RNA (rRNA). Tollervey explains how these small RNAs accomplish this targeting and discusses the possible role of this rRNA modification.

Pop3p is essential for the activity of the RNase MRP and RNase P ribonucleoproteins in vivo

RNase MRP is a ribonucleoprotein (RNP) particle which is involved in the processing of pre‐rRNA at site A3 in internal transcribed spacer 1. Although RNase MRP has been analysed functionally, the structure and composition of the particle are not well characterized. A genetic screen for mutants which are synthetically lethal (sl) with a temperature‐sensitive (ts) mutation in the RNA component of RNase MRP (rrp2‐1) identified an essential gene, POP3, which encodes a basic protein of 22.6 kDa predicted molecular weight. Overexpression of Pop3p fully suppresses the ts growth phenotype of the rrp2‐1 allele at 34°C and gives partial suppression at 37°C. Depletion of Pop3p in vivo results in a phenotype characteristic of the loss of RNase MRP activity; A3 cleavage is inhibited, leading to under‐accumulation of the short form of the 5.8S rRNA (5.8SS) and formation of an aberrant 5.8S rRNA precursor which is 5′‐extended to site A2. Pop3p depletion also inhibits pre‐tRNA processing; tRNA primary transcripts accumulate, as well as spliced but 5′‐ and 3′‐unprocessed pre‐tRNAs. The Pop3p depletion phenotype resembles those previously described for mutations in components of RNase MRP and RNase P (rrp2‐1, rpr1‐1 and pop1‐1). Immunoprecipitation of epitope‐tagged Pop3p co‐precipitates the RNA components of both RNase MRP and RNase P. Pop3p is, therefore, a common component of both RNPs and is required for their enzymatic functions in vivo. The ubiquitous RNase P RNP, which has a single protein component in Bacteria and Archaea, requires at least two protein subunits for its function in eukaryotic cells.

Two distinct recognition signals define the site of endonucleolytic cleavage at the 5'-end of yeast 18S rRNA.

Three of the four eukaryotic ribosomal RNA molecules (18S, 5.8S and 25–28S rRNA) are transcribed as a single precursor, which is subsequently processed into the mature species by a complex series of cleavage and modification reactions. Early cleavage at site A1 generates the mature 5′‐end of 18S rRNA. Mutational analyses have identified a number of upstream regions in the 5′ external transcribed spacer (5′ ETS), including a U3 binding site, which are required in cis for processing at A1. Nothing is known, however, about the requirement for cis‐acting elements which define the position of the 5′‐end of the 18S rRNA or of any other eukaryotic rRNA. We have introduced mutations around A1 and analyzed them in vivo in a genetic background where the mutant pre‐rRNA is the only species synthesized. The results indicate that the mature 5′‐end of 18S rRNA in yeast is identified by two partially independent recognition systems, both defining the same cleavage site. One mechanism identifies the site of cleavage at A1 in a sequence‐specific manner involving recognition of phylogenetically conserved nucleotides immediately upstream of A1 in the 5′ ETS. The second mechanism specifies the 5′‐end of 18S rRNA by spacing the A1 cleavage at a fixed distance of 3 nt from the 5′ stem‐loop/pseudoknot structure located within the mature sequence. The 5′ product of the A1 processing reaction can also be identified, showing that, in contrast to yeast 5.8S rRNA, the 5′‐end of 18S rRNA is generated by endonucleolytic cleavage.

U14 small nucleolar RNA makes multiple contacts with the pre-ribosomal RNA

The small nucleolar RNA (snoRNA) U14 has two regions of extended primary sequence complementarity to the 18S rRNA. The 3′ region (domain B) shows the consensus structure for the methylation guide class of snoRNAs, whereas base-pairing between the 5′ region (domain A) and the 18S rRNA sequence is required for the formation of functional ribosomes. Between domains A and B lies another essential region (domain Y). Here we report that yeast U14 can be cross-linked in vivo to the pre-rRNA; cross-linking is detected exclusively with the 35S primary transcript. Many nucleotides in U14 that lie outside of domains A and B are cross-linked to the pre-rRNA; in particular the essential domain Y region is cross-linked at several sites. U14 is, therefore, in far more extensive contact with the pre-rRNA than predicted from simple base-pairing models. Moreover, U14 can be cross-linked to other small RNA species. The functional interactions made by U14 during ribosome synthesis are likely to be very complex.