Deep Sequencing Shows Multiple Oligouridylations Are Required for 3′ to 5′ Degradation of Histone mRNAs on Polyribosomes

Histone mRNAs are rapidly degraded when DNA replication is inhibited during S-phase with degradation initiating with oligouridylation of the stemloop at the 3′ end. We developed a customized RNA-Seq strategy to identify the 3′ termini of degradation intermediates of histone mRNAs. Using this strategy, we identified two types of oligouridylated degradation intermediates: RNAs ending at different sites of the 3′ side of the stemloop that resulted from initial degradation by 3′hExo and intermediates near the stop codon and within the coding region. Sequencing of polyribosomal histone mRNAs revealed that degradation initiates and proceeds 3′ to 5′ on translating mRNA and many intermediates are capped. Knockdown of the exosome-associated exonuclease Pml/Scl-100, but not the Dis3L2 exonuclease, slows histone mRNA degradation, consistent with 3′ to 5′ degradation by the exosome containing PM/Scl-100. Knockdown of No-go decay factors also slowed histone mRNA degradation, suggesting a role in removing ribosomes from partially degraded mRNAs

The role of the cap and poly(A) tail in mRNA metabolism

Most eukaryotic mRNAs possess a 7-methyl-guanosine at their 5\u27 ends (5\u27 cap) and a poly(A) tail at their 3\u27 ends. These structures play important roles in gene expression, for example, it is believed that an mRNA must possess a poly(A) tail and possibly a 5\u27 cap to leave the nucleus and both of these structures play important roles in translation and mRNA stability. Much of what we know about the roles of the 5\u27 cap and 3\u27 poly(A) tail results from experiments using cell extracts, exogenous mRNA electroporated into cells, or analyses performed with very unhealthy mutant cells, all of which may not imitate in vivo conditions. To circumvent these problems, I have made reporter constructs that are transcribed in a wild type yeast cell and then cleaved, resulting in an unadenylated or uncapped cleavage product. I have demonstrated that 5\u27 unadenylated cleavage products can be translated but are rapidly targeted to the cytoplasmic exosome for degradation. In accordance with a 5\u27 cap being required for translation and mRNS stability, uncapped 3\u27 cleavage products are unstable and are not translated. This approach was then used to test the proposed requirement of the poly(A) tail in recognition of premature termination codons by the nonsense-mediated mRNA decay pathway. Contrary to what was believed, unadenylated nonsense mRNAs are still recognized as nonsense. This indicates that the distance between a premature termination codon and the poly(A) tail is not required for nonsense-mediated decay in yeast. Another set of studies has used reporter mRNAs that contain cleavage signals for the nuclear endonuclease, Rnt1p. These constructs have been used to study the requirement of the 5\u27 cap and poly(A) tail for mRNA export. Using these reporters, I have demonstrated that the 5\u27 cap and poly(A) tail are not required for export. This work has provided new insights into many aspects of gene expression. Furthermore, the constructs that I have described should be useful for more detailed studies of the roles of the 5\u27 cap and poly(A) tail in all aspects of mRNA metabolism

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

A Genomic Screen in Yeast Reveals Novel Aspects of Nonstop mRNA Metabolism

Nonstop mRNA decay, a specific mRNA surveillance pathway, rapidly degrades transcripts that lack in-frame stop codons. The cytoplasmic exosome, a complex of 3′–5′ exoribonucleases involved in RNA degradation and processing events, degrades nonstop transcripts. To further understand how nonstop mRNAs are recognized and degraded, we performed a genomewide screen for nonessential genes that are required for nonstop mRNA decay. We identified 16 genes that affect the expression of two different nonstop reporters. Most of these genes affected the stability of a nonstop mRNA reporter. Additionally, three mutations that affected nonstop gene expression without stabilizing nonstop mRNA levels implicated the proteasome. This finding not only suggested that the proteasome may degrade proteins encoded by nonstop mRNAs, but also supported previous observations that rapid decay of nonstop mRNAs cannot fully explain the lack of the encoded proteins. Further, we show that the proteasome and Ski7p affected expression of nonstop reporter genes independently of each other. In addition, our results implicate inositol 1,3,4,5,6-pentakisphosphate as an inhibitor of nonstop mRNA decay

Reporter mRNAs cleaved by Rnt1p are exported

and degraded in the cytoplas

Deep Sequencing Shows Multiple Oligouridylations Are Required for 3′ to 5′ Degradation of Histone mRNAs on Polyribosomes

Histone mRNAs are rapidly degraded when DNA replication is inhibited during S-phase with degradation initiating with oligouridylation of the stemloop at the 3′ end. We developed a customized RNA-Seq strategy to identify the 3′ termini of degradation intermediates of histone mRNAs. Using this strategy, we identified two types of oligouridylated degradation intermediates: RNAs ending at different sites of the 3′ side of the stemloop that resulted from initial degradation by 3′hExo and intermediates near the stop codon and within the coding region. Sequencing of polyribosomal histone mRNAs revealed that degradation initiates and proceeds 3′ to 5′ on translating mRNA and many intermediates are capped. Knockdown of the exosome-associated exonuclease Pml/Scl-100, but not the Dis3L2 exonuclease, slows histone mRNA degradation, consistent with 3′ to 5′ degradation by the exosome containing PM/Scl-100. Knockdown of No-go decay factors also slowed histone mRNA degradation, suggesting a role in removing ribosomes from partially degraded mRNAs