Making and breaking the message

A report on the Cold Spring Harbor Laboratory meeting 'Eukaryotic mRNA Processing', Cold Spring Harbor, USA, 20-24 August 2003

The Mtr4 Ratchet Helix and Arch Domain both Function to Promote RNA Unwinding

Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosomemiated 3-5 turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The ‘ratchet helix’ is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters in vitro activity in Mtr4 and TRAMP and causes slow growth phenotypes in vivo. We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates

A budding yeast model for human disease mutations in the EXOSC2 cap subunit of the RNA exosome complex

RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4 The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies

Conserved Functions of Yeast Genes Support the Duplication, Degeneration and Complementation Model for Gene Duplication

Gene duplication is often cited as a potential mechanism for the evolution of new traits, but this hypothesis has not been thoroughly tested experimentally. A classical model of gene duplication states that after gene duplication one copy of the gene preserves the ancestral function, while the other copy is free to evolve a new function. In an alternative duplication, divergence, and complementation model, duplicated genes are preserved because each copy of the gene loses some, but not all, of its functions through degenerating mutations. This results in the degenerating mutations in one gene being complemented by the other and vice versa. These two models make very different predictions about the function of the preduplication orthologs in closely related species. These predictions have been tested here for several duplicated yeast genes that appeared to be the leading candidates to fit the classical model. Surprisingly, the results show that duplicated genes are maintained because each copy carries out a subset of the conserved functions that were already present in the preduplication gene. Therefore, the results are not consistent with the classical model, but instead fit the duplication, divergence, and complementation model

FUNCTIONS OF DCP2 AND SKI7 IN MRNA DEGRADATION

Posttranscriptional gene regulation is essential to maintain gene expression fidelity. This is partially achieved by mRNA decay. When no longer required, mRNA is degraded by two alternative pathways. The decapping enzyme Dcp2 removes the 5` m7G cap of mRNAs, allowing Xrn1 to degrade the mRNA from the 5` end. Alternatively, mRNA is degraded from the 3` end by the RNA exosome. While decapping by Dcp2 is a critical step in mRNA decay, its physiological function has been unclear. Null mutations of Saccharomyces cerevisiae DCP2 have been reported to be lethal in some studies but slow-growing in others. In this study, I show that Dcp2 is required for continuous growth under standard laboratory conditions. I found multiple suppressors of the growth defect of a DCP2 null mutant via experimental evolution and genome sequencing. These suppressors act by at least three independent mechanisms. They do not rescue defects in mRNA decay. Instead, they appear to partially alleviate global disturbance of the transcriptome in the dcp2 mutant, which confers growth improvement. One of the suppressors appears to suppress by affecting translation of CUY codons. mRNAs that are generated by mistakes need to be degraded. Nonstop mRNAs are transcripts that lack an in-frame stop codon. Thus, the ribosome translating the nonstop mRNA reads through downstream of the coding region and produces aberrant nonstop proteins that are potentially toxic to cells. Cells exploit surveillance mechanisms to suppress the expression of nonstop mRNAs. The RNA exosome and its cofactors, the Ski complex and Ski7, are required for degradation of nonstop mRNAs. While Ski7 is known to have nonstop decay specific function, its exact role in nonstop decay remains unknown. Moreover, whether Ski7-mediated nonstop mRNA decay is mechanistically linked to the surveillance pathway for nonstop protein has been unclear. In this study, I determine the genetic interactions between components of the two surveillance pathways and show that the Ski7-mediated nonstop mRNA decay mechanism functions independently of nonstop protein degradation mechanism

Functions of the tRNA splicing endonuclease and other adventures in RNA processing

The tRNA splicing endonuclease (TSEN), has been studied for over three decades for its function in tRNA splicing. However, this enzyme has other functions that are just beginning to be characterized. Mutations in TSEN cause the neuronal disease pontocerebellar hypoplasia (PCH) that is characterized by atrophy of the cerebellum and pons, overall developmental failure, and usually results in death before adolescence. How mutations in TSEN cause these neuronal defects and disease is not understood. In yeast, TSEN has another essential function that is independent of tRNA splicing and is still unknown. In this thesis I strived to understand the other function of the TSEN complex. TSEN has one mRNA target in yeast which led me to the hypothesis that TSEN could cleave other mRNAs. I used Parallel Analysis of RNA Ends (PARE) to identify other mRNA substrates of TSEN. I found TSEN cleaves a subset of mRNAs that encode mitochondrial localized proteins. In vivo and in vitro analysis determine TSEN recognizes an A before its cleavage sites. We identified some sequence and localization requirements for TSEN targets but it is likely other factors play a role in substrate recognition such as structure of the mRNA target. Overall we used PARE to identify a novel endonuclease decay pathway, termed TED, in which TSEN can degrade a select group of mRNAs. Yeast genetic screens were used to complement our RNAseq approach to finding the other essential function of TSEN. A spontaneous suppressor screen identified mutations in Dbr1 as suppressors of only the other essential function a mutant sen2. Because mutations in Dbr1 could only complement a partially functional TSEN complex and the catalytic activity of Dbr1 must be lost for this suppression, we propose that Dbr1 and TSEN complete for a common substrate. Through RNAseq, we discovered that loss of the other essential function of TSEN triggers the Gcn4 response. This response is protective in our sen2 mutant and when Dbr1 is mutated in addition to sen2, the Gcn4 response is reduced as TSEN now has no competition for substrate to perform its essential function. As TSEN is involved in mRNA decay through the TED pathway, I wondered what enzymes could be involved in the degradation of these cleavage products. To investigate this, we used PARE to define targets of the exonuclease Dxo1 and the kinase Trl1. This revealed Dxo1 can “nibble” downstream of endonuclease cleavage and decapping but that its main function is in processing the 25S’ to the 25S rRNA in the cytoplasm. Trl1 can also act downstream of endonuclease decay in the TED pathway by phosphorylating the 5’ end of TSEN cleavage products. Though the other essential function of TSEN remains elusive, this research uncovered the participation of TSEN in mRNA decay and the functions of downstream enzymes as well as the identification of a potential competitor, Dbr1

The RNA Exosome Channeling and Direct Access Conformations Have Distinct In Vivo Functions

The RNA exosome is a 3′–5′ ribonuclease complex that is composed of nine core subunits and an essential catalytic subunit, Rrp44. Two distinct conformations of Rrp44 were revealed in previous structural studies, suggesting that Rrp44 may change its conformation to exert its function. In the channeling conformation, (Rrp44ch), RNA accesses the active site after traversing the central channel of the RNA exosome, whereas in the other conformation, (Rrp44da), RNA gains direct access to the active site. Here, we show that the Rrp44da exosome is important for nuclear function of the RNA exosome. Defects caused by disrupting the direct access conformation are distinct from those caused by channel-occluding mutations, indicating specific functions for each conformation. Our genetic analyses provide in vivo evidence that the RNA exosome employs a direct-access route to recruit specific substrates, indicating that the RNA exosome uses alternative conformations to act on different RNA substrates

Yeast transcripts cleaved by an internal ribozyme provide new insight into the role of the cap and poly(A) tail in translation and mRNA decay

It has been proposed that the 7-methylguanosine cap and poly(A) tail of mRNAs have important functions in translation and transcript stability. To directly test these roles of the cap and poly(A) tail, we have constructed plasmids with a ribozyme within the coding region or 3′ UTR of reporter genes. We show that the unadenylated 5′ cleavage product is translated and is rapidly degraded by the cytoplasmic exosome. This exosome-mediated decay is independent of the nonstop mRNA decay pathway, and, thus, reveals an additional substrate for exosome-mediated decay that may have physiological equivalents. The rapid decay of this transcript in the cytoplasm indicates that this unadenylated cleavage product is rapidly exported from the nucleus. We also show that this cleavage product is not subject to rapid decapping; thus, the lack of a poly(A) tail does not always trigger rapid decapping of the transcript. We show that the 3′ cleavage product is rapidly degraded by Xrn1p in the cytoplasm. We cannot detect any protein from this 3′ cleavage product, which supports previous data concluding that the 5′ cap is required for translation. The reporter genes we have utilized in these studies should be generally useful tools in studying the importance of the poly(A) tail and 5′ cap of a transcript for export, translation, mRNA decay, and other aspects of mRNA metabolism in viv