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

Abstract

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 39 UTR of reporter genes. We show that the unadenylated 59 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 39 cleavage product is rapidly degraded by Xrn1p in the cytoplasm. We cannot detect any protein from this 39 cleavage product, which supports previous data concluding that the 59 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 59 cap of a transcript for export, translation, mRNA decay, and other aspects of mRNA metabolism in vivo

Different nuclease requirements for exosome-mediated degradation of normal and nonstop mRNAs

“…For example, in no-go decay, mRNAs that have stalled ribosomes within their coding region are cleaved by an unknown endonuclease (25,29). Similar cleavage products can be generated by inserting a ribozyme into an mRNA (32). Though these pathways are initiated by a variety of endonucleases, the fragments resulting from this cleavage are degraded by common exonucleases.…”

“…Though these pathways are initiated by a variety of endonucleases, the fragments resulting from this cleavage are degraded by common exonucleases. Specifically, the 5′ fragments are degraded by the cytoplasmic exosome, and the 3′ fragments are degraded by the 5′-to-3′ exonuclease Xrn1p (25)(26)(27)(28)(32)(33)(34).…”

“…To determine whether the nuclease requirements for the degradation of other aberrant transcripts were similar to that of nonstop mRNAs, we analyzed the degradation of a ribozyme-cleaved transcript. Previous studies indicate that the 5′ cleavage product resulting from ribozyme cleavage is rapidly degraded by the cytoplasmic exosome (32). To determine whether the nuclease activities of Rrp44p were needed for this mRNA decay pathway, a plasmid containing a hammerhead ribozyme inserted into the HIS3 gene (his3-Rz) was transformed into the rrp44-endo − or rrp44-exo − mutants.…”

A conserved role for cytoplasmic poly(A)-binding protein 1 (PABPC1) in nonsense-mediated mRNA decay

“…3 0 UTRs specifying rapid decay confer immunity to NMD The results described so far show that at least three different classes of nonsense mRNAs escape NMD: (1) ribozymeterminated mRNAs; (2) unadenylated mRNAs whose 3 0 ends are generated by the histone 3 0 -end-processing machinery; and (3) mRNAs with a 3 0 UTR derived from hsp70. In S. cerevisiae, ribozyme-terminated mRNAs are rapidly degraded by the cytoplasmic exosome and/or XRN1 (Dower et al, 2004;Meaux and van Hoof, 2006). Moreover, the hsp70 3 0 UTR contain several AU-rich elements that promote rapid decay.…”

Regulation of the Nuclear Poly(A)-binding Protein by Arginine Methylation in Fission Yeast

“…Whereas numerous studies indicate that the presence of a poly(A) tail on a eukaryotic mRNA is required for efficient nuclear export, RNA stability, and translational control (1), recent findings have questioned the direct role of the poly(A) tail in specific steps of mRNA metabolism (2)(3)(4). The length of this stretch of polyadenosines is controlled in a species-dependent manner with average lengths of 70 and 250 nucleotides in yeast and mammals, respectively (5,6).…”