Role of Polyadenylation in Nucleocytoplasmic Transport of mRNA

Abstract

To examine the role of polyadenylation in the nuclear export of mRNA, we have replaced the poly(A) signal in a Rev-responsive human immunodeficiency virus type 1-based reporter gene with a cis-acting hammerhead ribozyme. Transcripts from this gene thus acquire a 3 terminus by cis-ribozyme cleavage rather than by polyadenylation. The nuclear and cytoplasmic distribution of transcripts was investigated using transient gene expression and quantitative RNase protection assays. In the absence of Rev, a basal level of polyadenylated unspliced mRNA transcribed from a poly(A) signal-containing control reporter gene was detected in the cytoplasm of transfected COS7 cells. However, cytoplasmic ribozyme-cleaved unspliced RNA was only barely detectable. The nuclear/cytoplasmic (n/c) ratio of polyadenylated RNAs was 3.8, while the n/c ratio for ribozyme cis-cleaved RNAs was 33. The cytoplasmic localization of the polyadenylated unspliced mRNA was enhanced about 10-fold in the presence of Rev and the Rev-responsive element. In marked contrast to this, ribozymecleaved RNA accumulated almost exclusively (n/c ratio of 28) in the nucleus in the presence of Rev. Actinomycin D time course analysis suggested that the low levels of the cytoplasmic ribozyme-cleaved RNAs in both the presence and absence of Rev were due to severe export deficiency of ribozyme-cleaved RNA. Finally, by inserting a 90-nucleotide poly(A) stretch directly upstream of the ribozyme cassette, we have demonstrated that a long stretch of poly(A) near the 3 end of a ribozyme-cleaved transcript is not sufficient for directing mRNA export. Taken together, these results suggest that polyadenylation is required for the nucleocytoplasmic transport of mRNA and that Rev interaction with the Rev-responsive element cannot bypass this requirement.Messenger RNA precursors (pre-mRNAs) are synthesized in the nucleus by RNA polymerase II and then subjected to a series of processing reactions which include the addition of a 7-methylguanosine cap at the 5Ј end, the removal of introns by splicing, and the generation of mature 3Ј ends. With the exception of histone genes, whose transcripts end in a highly conserved stem-loop structure (for reviews see references 2 and 36), the 3Ј end is formed by cleavage and polyadenylation (recently reviewed; see reference 47). Mature RNA molecules are then translocated to the cytoplasm through the nuclear pore complex (see reference 12 for review). The mechanism of mRNA nuclear export is poorly understood, although this process is fundamental to eukaryotic gene expression. Recently, however, evidence has begun to accumulate that capping and subsequent processing of pre-mRNA to generate mature mRNA influence the efficiency of nuclear export (reviewed in references 12, 20, and 26).Evidence that the 5Ј monomethylguanosine cap structure may serve as a positive signal for mRNA nuclear export was derived from microinjection experiments carried out with Xenopus oocytes (9, 18, 21). It was shown that mRNAs carrying the normal 5Ј cap structure were rapid...

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation

“…On the other hand, the stabilization of initially polyadenylated and of ribozyme-cleaved mRNAs might occur through overlapping, but distinct, mechanisms. For example, Pab1p might be required for transport of the ribozyme-generated mRNA from the nucleus (Huang and Carmichael 1996). Escape from the nucleus might then permit stabilization.…”

The Poly(A) Tail Inhibits the Assembly of a 3′-to-5′ Exonuclease in an In Vitro RNA Stability System

“…The tail is a dynamic structure which serves as a mediator through which several important cellular processes including the initiation of translation (31), nucleocytoplasmic transport (18), and mRNA stability (7), are regulated. Most mRNAs are rapidly degraded following deadenylation of their 3Ј ends to approximately 30 adenylate residues (8,21,27,34).…”

The Polyadenylation of RNA in Plants

“…A growing body of evidence indicates that mRNA polyadenylation may be physically linked to the processes of intron remova1 (e.g. Niwa et al, 1990) and transcription termination (McCracken et al, 1997), and other studies imply an interplay between polyadenylation and transport of mRNA (Huang and Carmichael, 1996). Thus, it may be more appropriate to consider mRNA 3'-end formation as part of a larger series of events that begins with the initiation of transcription by RNA polymerase I1 and ends with the delivery of a mature, polyadenylated mRNA to the cytoplasm.…”