3′-End processing of pre-mRNA in eukaryotes

Abstract

Abstract3P-Ends of almost all eukaryotic mRNAs are generated by endonucleolytic cleavage and addition of a poly(A) tail. In mammalian cells, the reaction depends on the sequence AAUAAA upstream of the cleavage site, a degenerate GU-rich sequence element downstream of the cleavage site and stimulatory sequences upstream of AAUAAA. Six factors have been identified that carry out the two reactions. With a single exception, they have been purified to homogeneity and cDNAs for 11 subunits have been cloned. Some of the cooperative RNA-protein and protein-protein interactions within the processing complex have been analyzed, but many details, including the identity of the endonuclease, remain unknown. Several examples of regulated polyadenylation are being analyzed at the molecular level. In the yeast Saccharomyces cerevisiae, sequences directing cleavage and polyadenylation are more degenerate than in metazoans, and a downstream element has not been identified. The list of processing factors may be complete now with approximately a dozen polypeptides, but their functions in the reaction are largely unknown. 3P-Processing is known to be coupled to transcription. This connection is thought to involve interactions of processing factors with the mRNA cap as well as with RNA polymerase II. ß

Retinoid‐dependent mRNA expression and poly‐(A) contents in bovine oocytes meiotically arrested and/or matured in vitro

“…However, it is not surprising that increased gene expression occurs with enhanced mRNA processing, since proteins that catalyze polyadenylation are associated with RNA polymerase II (Pol II), providing a link between transcription and formation of the 3 0 end of mRNA (Maniatis and Reed, 2000;Proudfoot et al, 2000). A functional polyadenylation signal terminates transcription and release Pol II (Whale and Ruegsegger, 1999;Shatkin and Manley, 2000), although polyadenylation seems to continue post-transcriptionally (Osheim et al, 1999(Osheim et al, , 2002.…”

Protein factors in pre-mRNA 3′-end processing

“…The DSE is less conserved and more diffuse than the PAS [2,19]. The presence of this element was suggested by the observation that a deletion more than 10 nucleotides downstream of the cleavage site reduced polyadenylation three-fold [42].…”

“…Mutation of the PE forces cleavage to occur at unspecific locations in the 3′ untranslated region [19].…”

Replacement of the yeast TRP4 3??? untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail

“…The 39 untranslated region (39 UTR) of an mRNA plays an important role in 39 end processing of eukaryotic transcripts+ Sequence motifs, located in the 39 UTR, are necessary for cleavage at a specific site and the addition of a poly(A) tail (for reviews, see Wahle & Rüegsegger, 1999;Zhao et al+, 1999)+ The sequence motifs of yeast transcripts that direct cleavage and polyadenylation are not as conserved as in higher eukaryotes+ Several yeast 39 untranslated regions were shown to carry degenerate motifs that can at least partially replace one another (Egli et al+, 1995;Guo et al+, 1995)+ Deletion of the 39 UTR usually results in a significant reduction of the amount of gene product+ A deletion of the 39 UTR of the TRP4 gene led to a reduced amount of gene product without affecting cellular growth rates (Düvel et al+, 1999)+ A large multiprotein complex performs cleavage and polyadenylation of pre-mRNAs in eukaryotes (Keller & Minvielle-Sebastia, 1997;Zhao et al+, 1999)+ A subset of these factors affects only the polyadenylation efficiency whereas others are necessary for both cleavage and polyadenylation+ A complex pattern of interactions connects the different components with each other and cooperative interactions between the processing factors have been observed (Kessler et al+, 1997)+ As a consequence, cleavage and polyadenylation are coupled processes in vivo+ However, an uncoupling of cleavage and polyadenylation was achieved in vivo in yeast by using an artificial hammerhead ribozyme as a mRNA 39 end processing signal (Egli & Braus, 1994)+ Ribozymes are RNAs with an enzymatic activity for RNA cleavage (Altman, 1990;Cech, 1990)+ Hammerhead ribozymes are a subset of self-cleaving ribozymes that were originally isolated from plant viroids (Symons, 1992)+ Hammerhead ribozymes consist of three stem-loop-forming regions and two highly conserved single-stranded sequences+ Cleavage occurs at a triplet sequence, usually GUC+ Hydrolysis of the phosphodiester bond results in a 29-39-cyclic phosphate and a 59-hydroxyl product (Tanner, 1999)+ This is in contrast to enzymatic cleavage by processing factors that generally result in a 39-hydroxyl and a 59-phosphate+ Because the catalytic action of ribozymes has been studied thoroughly, new ribozymes were constructed providing applications in biotechnology and medicine+ Trans-acting ribozymes, for example, cleave target mRNAs at specific cleavage motifs, and are widely used to decrease the amount of the mRNA of genes of interest (Birikh et al+, 1997;Tanner, 1999)+ The hammerhead ribozyme used in this study cleaves itself efficiently in yeast (Egli & Braus, 1994)+ This prompted us to determine whether a hammerhead ribozyme could replace the normal mechanism of 39 end formation in an authentic yeast transcript+ We have now found that replacing the 39 UTR of the TRP4 gene with a hammerhead ribozyme results in a ...…”