A synthetic A tail rescues yeast nuclear accumulation of a ribozyme-terminated transcript

Abstract

To investigate the role of 3 end formation in yeast mRNA export, we replaced the mRNA cleavage and polyadenylation signal with a self-cleaving hammerhead ribozyme element. The resulting RNA is unadenylated and accumulates near its site of synthesis. Nonetheless, a significant fraction of this RNA reaches the cytoplasm. Nuclear accumulation was relieved by insertion of a stretch of DNA-encoded adenosine residues immediately upstream of the ribozyme element (a synthetic A tail). This indicates that a 3 stretch of adenosines can promote export, independently of cleavage and polyadenylation. We further show that a synthetic A tail-containing RNA is unaffected in 3 end formation mutant strains, in which a normally cleaved and polyadenylated RNA accumulates within nuclei. Our results support a model in which a polyA tail contributes to efficient mRNA progression away from the gene, most likely through the action of the yeast polyA-tail binding protein Pab1p.

Recognition of polyadenosine RNA by zinc finger proteins

“…Transcripts lacking poly(A) tails are not properly translated and are quickly degraded (40). Additionally, S. cerevisiae transcripts that are terminated by a self-cleaving ribozyme element and thereby lack poly(A) tails are at least partially retained in the nucleus (41), suggesting that the poly(A) tail may also play a role in mRNA export from the nucleus. By regulating the stability, translatability, and even subcellular localization of mRNA transcripts, the poly(A) tail and its associated proteins can act as potent posttranscriptional regulators of gene expression.…”

mRNA stability in the nucleus

“…More interestingly, it seems that the poly(A) tail itself is required for RNA release from the transcription site by using a self-cleaving hammerhead ribozyme to eliminate normal polyadenylation. The efficient RNA release of the non-poly(A) tail mRNA occurred only if an artificial poly(A) tail was introduced at the upstream of the ribozyme cleavage site (Dower et al, 2004). Also, deletion of PAB1, which encodes poly(A) binding protein, shuttled between cytoplasm and nucleus, and PAN, which encodes poly(A) ribonuclease to trim the poly(A) tail to a proper length for mRNA export, caused the exosome-dependent nucleus retention (Dunn et al, 2005).…”

PolydA and polyrA self‐structured by a europium and amino acid complex

“…All mRNAs have a number of adenylate residues at their 3 0 -end, the polyrA tail. The long polyrA tail is an important determinant of mRNA stability and maturation, and is essential for the initiation of translation [16,17]. PolyrA polymerase (PAP) catalyzes 3 0 -end polyrA synthesis, participates in an endonucleolytic cleavage step, and is one key factor in the polyadenylation of the 3 0 -end of mRNA.…”