Assembly of an Export-Competent mRNP Is Needed for Efficient Release of the 3′-End Processing Complex after Polyadenylation

Abstract

Before polyadenylated mRNA is exported from the nucleus, the 3-end processing complex is removed by a poorly described mechanism. In this study, we asked whether factors involved in mRNP maturation and export are also required for disassembly of the cleavage and polyadenylation complex. An RNA immunoprecipitation assay monitoring the amount of the cleavage factor (CF) IA component Rna15p associated with poly(A) ؉ RNA reveals defective removal of Rna15p in mutants of the nuclear export receptor Mex67p as well as other factors important for assembly of an export-competent mRNP. In contrast, Rna15p is not retained in mutants of export factors that function primarily on the cytoplasmic side of the nuclear pore. Consistent with a functional interaction between Mex67p and the 3-end processing complex, a mex67 mutant accumulates unprocessed SSA4 transcripts and exhibits a severe growth defect when this mutation is combined with mutation of Rna15p or another CF IA subunit, Rna14p. RNAs that become processed in a mex67 mutant have longer poly(A) tails both in vivo and in vitro. This influence of Mex67p on 3-end processing is conserved, as depletion of its human homolog, TAP/NXF1, triggers mRNA hyperadenylation. Our results indicate a function for nuclear mRNP assembly factors in releasing the 3-end processing complex once polyadenylation is complete.A significant advance in the area of eukaryotic gene expression has been the appreciation of multiple interrelationships between activities needed to get a functional mRNA to the cytoplasm for translation. Coordination occurs at the level of transcription, capping, splicing, 3Ј-end processing, and assembly of the mRNA into a ribonucleoprotein particle (RNP) that can be transported (for reviews, see references 2, 5, 12, 43, 51, and 55). Overseeing all of this is a nuclear mRNA surveillance mechanism which ensures that only correctly formed mRNP reaches the cytoplasm and that defective ones are degraded (for a review, see reference 70).Processing at the mRNA 3Ј end is accomplished by a multisubunit complex that recognizes signals on the nascent RNA, cleaves at the poly(A) site, and adds a poly(A) tract. The subunits of the complex are largely conserved across eukaryotes, and in the yeast Saccharomyces cerevisiae, this complex can be separated biochemically into three factors: cleavage and polyadenylation factor (CPF), cleavage factor (CF) IA, and Hrp1p (59). The length of the RNA poly(A) tail is restricted by the recruitment of specific poly(A)-binding proteins, such as Nab2p and Pab1p (6,19,22,40,80), and by the action of poly(A)-specific nucleases in the nucleus and the cytoplasm (59). Previous studies showed that 3Ј-end processing factors, with the exception of Hrp1p, Pab1p, and Nab2p, do not shuttle in and out of the nucleus (9, 38, 48). Thus, the exported mRNP does not include CF IA and CPF components, yet the signal sequences that stably hold these factors onto the mRNA as it receives its tail are present on the final polyadenylated product. Therefore, a mechanism must ex...

The Spt5 C-Terminal Region Recruits Yeast 3′ RNA Cleavage Factor I

“…In eukaryotes, transcription and translation take place in different cellular compartments, and any coupling between these processes would likely occur via the mRNA that exits the nucleus (27,50). Our data indicate that the CTR of Spt5 contributes to the coordination of transcription with 3= RNA processing, which in turn is coupled to mRNA export (29,46,72). Since the CTR occurs only in eukaryotic Spt5 homologs, it is likely that it emerged during evolution to maintain coupling between transcription and translation after the spatial separation of these processes.…”

Crossing the borders: Poly(A)-binding proteins working on both sides of the fence

“…41 Interestingly, this robust mRNA export defect was not accompanied by mRNA hyperadenylation, a phenotype that was previously associated with mRNA export defects in yeast, fruit flies and mammalian cells. 7,29,42,43 In Drosophila, specific pabp2 mutants also show shorter poly(A) tails using total RNA or individual mRNAs. 39 Although this remains unclear, this hypoadenylation phenotype is assumed to be linked to PABP2's nuclear role since an hyperadenylation phenotype is associated with the cytosolic function of Drosophila PABP2 (discussed above).…”

Rrp6, Rrp47 and Cofactors of the Nuclear Exosome

“…[27][28][29] The mechanistic details of the connection between the THO/Sub2 complex and the nuclear exosome remain unclear, but recent evidence suggests that THO/Sub2 defects inhibit the activity of the mRNA 3' end processing machinery. 29,30 One interpretation of these findings is that disruption of the transition from pre-mRNA to an export competent RNP may feedback to down regulate mRNA 3' end processing, thereby exposing transcripts to degradation by Exo11.…”