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 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 vivo