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• W2000371296 abstract "One of the most interesting formats for a scientific meeting involves bringing together researchers working in overlapping or closely related fields where the opportunity can arise for cross-fertilization and exchange of ideas. This was the aim of the recent workshop on mRNA processing, transport, and translation held at the Juan March Foundation in Madrid on March 11–13. All the fields represented shared a common interest in studying the synthesis, maturation, transport, stability, and function of mRNA. The presentations included talks on characterizing the machineries involved in splicing, polyadenylating, and translating mRNA; how RNAs and proteins are transported into and out of the nucleus; how posttranslational mechanisms can control gene expression; and how RNA mechanisms are involved in virus–host interactions. The venue was ideal, our hosts were generous and helpful, and the outcome was a tremendously stimulating meeting where every talk was followed by an in-depth, open discussion, usually lasting 10–15 minutes. Many of these discussions resumed in the evenings over Tapas and, as it is the Spanish custom to dine late, continued into the small hours. We hope that the Juan March Foundation will decide to stage a repeat performance in the future. Spliceosomes are the large RNA–protein complexes that catalyze excision of introns from mRNA precursors (pre-mRNA) in the nucleus. Introns are spliced by a two-step mechanism, both steps being transesterification reactions (reviewed by55Moore, J.M., Query, C.C., and Sharp, P.A. (1993). Splicing of precursors to mRNAs by the spliceosome. In The RNA World, R.F. Gesteland and J.F. Atkins, eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), pp. 303–358.Google Scholar). First, cleavage occurs at the 5′ intron–exon junction, generating a free 5′ exon and a branched intron–3′ exon as reaction intermediates. The branched structure is formed by the esterification of the 5′ end of the intron to a 2′ hydroxyl group of an adenosine residue (branch site), close to the 3′ end of the intron. The second step produces ligated exons (mRNA) and a fully excised branched intron as the reaction products. The major subunits of spliceosomes are the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs), which assemble on pre-mRNAs in a stepwise pathway along with additional protein splicing factors. Recognition of introns involves both protein–RNA and RNA–RNA interactions being made by splicing factors with conserved sequence elements on the pre-mRNA at the 5′ and 3′ intron–exon junctions and at the branch site (reviewed by55Moore, J.M., Query, C.C., and Sharp, P.A. (1993). Splicing of precursors to mRNAs by the spliceosome. In The RNA World, R.F. Gesteland and J.F. Atkins, eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), pp. 303–358.Google Scholar, 59Nilsen T.W RNA–RNA interactions in the spliceosome unravelling the ties that bind.Cell. 1994; 78: 1-4Abstract Full Text PDF PubMed Scopus (164) Google Scholar). Considerable effort is currently being directed toward identifying the protein components of spliceosomes, determining how they interact with pre-mRNA and with each other during the splicing reaction, and analyzing how these interactions are regulated. B. Seraphin (EMBL, Heidelberg) and P. Legrain (Institut Pasteur, Paris) reported studies on how 5′ splice sites and branch sites in budding yeast pre-mRNAs are recognized by splicing factors. Legrain described an exhaustive mutagenesis of the branch point consensus sequence, analyzed using an in vivo assay system, which showed that certain base substitutions could differentially affect the splicing of pre-mRNA and its transport out of the nucleus. Although base pairing at the branch point between the pre-mRNA and U2 snRNA is essential for splicing, it is apparently not linked to retention of unspliced pre-mRNA in the nucleus. Legrain reported that mutational data also point to an important RNA–protein interaction taking place at the nucleotide immediately upstream of the branch site consensus, where a U residue is moderately conserved. B. Seraphin (EMBL, Heidelberg) described how the mechanism for selecting the precise site of cleavage at the 5′ intron–exon junction involves a complex network of snRNA–pre-mRNA base pairing. First, the region to be cleaved is selected by base pairing between the 5′ terminus of U1 snRNA and the pre-mRNA. Subsequently, both U5 and U6 snRNAs participate in selecting the exact bond to be cleaved by base pairing to the pre-mRNA. Seraphin pointed out that there may be some redundancy in this mechanism, as certain point mutations at the 5′ splice site that alter splice site choice can be suppressed by introducing mutations into either U5 or U6 snRNAs that affect their potential base pairing interactions with the pre-mRNA. Splicing snRNPs are associated with a common complex of eight proteins, called Sm proteins, that bind directly to U1, U2, U4, and U5 snRNAs but not to U6. A conserved “Sm domain” has been identified at the amino termini of Sm proteins and used to identify new members of the Sm protein family in the sequence database (B. Seraphin). In yeast, 15 Sm-type proteins have been identified and several of these have been shown to associate with snRNAs in vivo. Interestingly, most of the Sm-type proteins can be subdivided into two groups that appear to form separate complexes. One complex corresponds to the previously known set of Sm proteins that bind to U1, U2, U4, and U5 snRNAs, while the other corresponds to a new complex that binds to U6 snRNA (B. Seraphin). Another advance facilitated by the burgeoning sequence databases is the isolation of a human homolog of the yeast splicing factor prp18, which was identified and cloned using information derived from nematode and rice EST sequences (A. Krainer, Cold Spring Harbor). Like its yeast counterpart, human prp18 protein is required for the second catalytic step of the splicing reaction. Either the yeast or human prp18 proteins will complement a HeLa splicing extract that has been immunodepleted of endogenous prp18, although the human gene will not complement a yeast prp18 mutant in vivo (A. Krainer). A conserved family of protein splicing factors found in Drosophila and human cells share a dipeptide repeat motif consisting of alternating serine and arginine residues (reviewed by20Fu X.-D The superfamily of arginine/serine-rich splicing factors.RNA. 1995; 1: 663-680PubMed Google Scholar). These “SR” proteins are involved in both constitutive and alternative splicing mechanisms and the family can be subdivided into two groups, typified respectively by the proteins SF2/ASF and SC35 (A. Krainer). These two groups of proteins differ in their substrate specificity in constitutive splicing and in their mode of activating alternative splicing. Krainer reported that the analysis of chimeric proteins, formed by swapping domains from SF2/ASF and SC35, has shown that the substrate specificities of the proteins correlate with the presence of specific constituent domains, particularly the RNP motif RNA-binding domains (RNA recognition motif). However, because different SR protein modules appear to be important with different substrates, Krainer noted that this analysis has not so far uncovered any simple rules for predicting the substrate specificity of, or the alternative splicing patterns mediated by, a particular SR protein. J. Valcarcel (EMBL, Heidelberg) presented new data on the function of U2AF, an essential, conserved splicing factor that also has SR dipeptide repeats but differs in its properties from SR proteins such as ASF/SF2 and SC-35. U2AF has two subunits, both containing SR repeat motifs, and binds directly to the polypyrimidine tract of pre-mRNAs through RNA binding domains in the large subunit, which do not require the SR motif to bind RNA. However, the SR repeats in the large subunit are required to stabilize the binding of U2 snRNP at the branch point and mutagenesis shows that (SR)7 is the minimum functional unit of the motif. Valcarcel reported that the arginine can be substituted by lysine but not by alanine, while the serine can be changed to glycine, methionine, or threonine without loss of function but cannot be replaced by negatively charged amino acids. The run of positive charges, which directly contacts the pre-mRNA at the branch site, may stabilize base pairing between the branch site and U2 snRNA. A particularly exciting new development was the report from J. Steitz (HHMI, Yale Medical School, New Haven) showing that a novel form of mammalian spliceosome containing U5 snRNP and the minor snRNPs U11 and U12, but not U1, U2, or U4/U6, assemble in vitro on a minor class of introns (called AT-AC introns) with noncanonical consensus splice sites (78Tarn W.-Y Steitz J A novel spliceosome containing U11, U12, and U5 snRNPs excises a minor class (AT-AC) intron in vitro.Cell. 1996; 84: 801-811Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). This confirms an earlier prediction by Hall and Padgett that U11 and U12 snRNPs may substitute for U1 and U2 snRNPs during splicing of AT-AC introns (29Hall S.L Padgett R.A Conserved sequences in a class of rare eukaryotic nuclear introns with non-consensus splice sites.J. Mol. Biol. 1994; 239: 357-365Crossref PubMed Scopus (181) Google Scholar). Recent studies by the same authors have also shown that U12 snRNA base pairs with the branch point of AT-AC introns in vivo (30Hall S.L Padgett R.A Requirement for U12 snRNA for in vivo splicing of a minor class of eukaryotic nuclear pre-mRNA introns.Science. 1996; 271: 1716-1718Crossref PubMed Scopus (164) Google Scholar). Steitz reported that U5 snRNP function is required for splicing both classes of introns, while U1 and U2 snRNPs are not required for splicing AT-AC introns. Conversely, U11 and U12 appear only involved in splicing ATAC introns. A major question that remains is whether other minor snRNAs are also in the ATAC spliceosomes and playing the role of U4/U6 snRNP. It is also unclear just how many protein splicing factors will be shared by the two forms of spliceosome. Spliceosomes purified from extracts containing two distinct forms of U1 snRNA, which differ in the sequence of their loop B regions, were reported to contain disproportionately more of the U1b variant (M. Bach, Barcelona, Spain). It therefore appears that spliceosome structure may be more heterogeneous than was previously thought. A. Lamond (University of Dundee, Scotland) reported a collaborative project by the groups of Lamond and Mann to systematically identify all the proteins in the mammalian spliceosome. This project takes advantage of the powerful new developments in nanoelectrospray mass spectrometry (84Wilm M Shevchenko A Houthaeve T Breit S Schweigerer L Fotsis T Mann M Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.Nature. 1996; 379: 466-469Crossref PubMed Scopus (1490) Google Scholar), which facilitate mass and sequence analysis of purified proteins in levels as low as 0.1 pmol. In combination with the rapidly expanding database of EST clones, it is hoped that this approach will accelerate the characterization of new splicing factors and make the clones available for future detailed functional studies (A. Lamond). By far the most lively discussion at the meeting was stimulated by the astonishing presentation from C. Milstein (MRC, Cambridge, England), who addressed the mechanism whereby B cells eliminate mRNAs containing premature stop codons. These stop codons arise as a result of the DNA rearrangements that occur when B cells mature in response to antigen. Igκ light chain mRNAs containing premature stop codons are expressed at very low levels (45Lozano F Maertzdorf B Pannell R Milstein C Low cytoplasmic mRNA levels of immunoglobulin kappa light chain genes containing nonsense codons correlate with inefficient splicing.EMBO J. 1994; 13: 4617-4622Crossref PubMed Scopus (81) Google Scholar). The mechanism responsible for this effect appears to take place in the nucleus and involve an inhibition of splicing of κ light chain pre-mRNAs containing premature stop codons (2Aoufouchi S Yélamos J Milstein C Nonsense mutations inhibit RNA splicing in a cell free system recognition of mutant codon is independent of protein synthesis and tissue specific.Cell. 1996; 85: 415-422Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Milstein reported that this effect is both cell type– and gene-specific and could be reproduced in a soluble S100 system, which would appear to exclude the involvement of intact ribosomes or feedback from the translation system to the spliceosome. How do B cells screen pre-mRNAs in the nucleus for in frame stop codons? Answering this question is clearly going to be a major focus of future work. W. Keller (Biozentrum, Basel, Switzerland) reported on progress in studying the mechanism of polyadenylation of nuclear pre-mRNA. This involves a two-step mechanism in which the substrate is first cleaved downstream of the conserved AAUAAA motif, then a tail of 150–250 adenosine residues is polymerized onto the free 3′ terminus. Addition of the poly(A) tail involves an initial oligoadenylation at the 3′ end of the cleaved mRNA followed by binding of a protein called PABII and subsequent extension of the tail (reviewed by36Keller W No end yet to messenger RNA 3′ processing!.Cell. 1995; 81: 829-832Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 81Wahle E Keller W The biochemistry of polyadenylation.Trends Biochem. Sci., in press. 1996; Google Scholar). Considerable advances have been made in cloning and characterizing the components of the polyadenylation machineries from both mammals and yeast and numerous homologs have been identified in the two systems (W. Keller). The polyadenylation machinery is large and surprisingly complex, including factors specifically involved in either cleavage or poly(A) addition and factors required for both steps. CPSF is a mammalian factor required for both steps that has a subunit of 160 kDa which binds directly to the AAUAAA motif. CPSF and poly(A) polymerase (PAP) are sufficient in vitro to polyadenylate a precleaved substrate containing the AAUAAA motif. Keller reported that the catalytic core of PAP lies at the amino terminus and contains at least three essential aspartate residues that, if mutated, severely reduce Kcat for polyadenylation. The catalytic domain of PAP shows similarity to the catalytic domains of other nucleotidyltransferases, notably tRNA CCA adding enzyme, DNA terminal transferase and DNA polymerase β (47Martin G Keller W Mutational analysis of mammalian poly(A) polymerase identifies a region for primer binding and a catalytic domain, homologous to the family X polymerases, and to other nucleotidyltransferases.EMBO J., in press. 1996; Google Scholar). The nucleus is a highly structured organelle with many nuclear antigens concentrated in specific subnuclear domains. The groups of A. Lamond and G. Dreyfuss (HHMI, University of Pennsylvania, Philadelphia) presented new studies on distinct classes of subnuclear domains called, respectively, coiled bodies and gems (see Figure 1). Coiled bodies contain splicing snRNPs, but not the SR family of protein splicing factors, and are found either closely associated with the periphery of the nucleolus or free in the nucleoplasm (reviewed by4Bohmann K Ferreira J Santama N Weis K Lamond A.I Molecular analysis of the coiled body.J. Cell Science (Suppl.). 1995; 19: 107-113Crossref PubMed Google Scholar). Lamond reported a combination of mutational data and studies with phosphatase inhibitors showing that coiled bodies can be induced to form within nucleoli and suggested that trafficking of coiled bodies between the nucleolus and nucleoplasm may be controlled by a protein phosphorylation mechanism. A new class of subnuclear body has been identified that contains the protein encoded by the survival of motor neuron (SMN) gene (G. Dreyfuss). This has been identified as the determining gene for spinal muscular atrophy (SMA), a fatal autosomal recessive disease causing progressive muscular wasting and paralysis (41Lefebvre S Burglen L Reboullet S Clermont O Burlet P Viollet L Benichou B Cruaud C Millasseau P Zeviani M et al.Identification and characterization of a spinal muscular atrophy-determining gene.Cell. 1995; 80: 155-165Abstract Full Text PDF PubMed Scopus (2744) Google Scholar). Dreyfuss described how the SMN protein is specifically localized in subnuclear bodies that are similar to coiled bodies in size and number, but are distinct structures that do not contain splicing snRNPs. They have been called gems, for Gemini of coiled bodies (44Liu Q Dreyfuss G A novel structure containing the survival of motor neurons protein.EMBO J., in press. 1996; Google Scholar). Future studies will be aimed at discovering the precise biolgical functions of coiled bodies, gems, and other classes of subnuclear bodies in which specific nuclear antigens concentrate. Heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins are abundant pre-/mRNA-binding proteins that bind to nascent transcripts, influence their interactions with trans-acting factors including spliceosomal snRNPs, and remain associated with the processed nuclear mRNAs (reviewed by14Dreyfuss G Matunis M.J Pĩol-Roma S Burd C.G hnRNP proteins and the biogenesis of mRNA.Annu. Rev. Biochem. 1993; 62: 289-321Crossref PubMed Scopus (1292) Google Scholar). Several observations suggest that hnRNP proteins also have a role in the export of mRNAs from the nucleus to the cytoplasm. First, several of the abundant hnRNP proteins, including A1, A2, D, I, and K, shuttle continuously and rapidly between the nucleus and the cytoplasm (64Piñol-Roma S Dreyfuss G Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm.Nature. 1992; 355: 730-732Crossref PubMed Scopus (720) Google Scholar65Piñol-Roma S Dreyfuss G hnRNP proteins localization and transport between the nucleus and the cytoplasm.Trends Cell Biol. 1993; 3: 151-155Abstract Full Text PDF PubMed Scopus (127) Google Scholar). Second, the shuttling hnRNP proteins, specifically A1, are associated with mRNA both in the nucleus and in the cytoplasm (64Piñol-Roma S Dreyfuss G Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm.Nature. 1992; 355: 730-732Crossref PubMed Scopus (720) Google Scholar65Piñol-Roma S Dreyfuss G hnRNP proteins localization and transport between the nucleus and the cytoplasm.Trends Cell Biol. 1993; 3: 151-155Abstract Full Text PDF PubMed Scopus (127) Google Scholar); their association with cytoplasmic mRNAs is brief, as they normally return to the nucleus rapidly. Third, an A1 homolog has been observed to be associated with a specific mRNA, the Balbiani ring mRNA, as it is being translocated to the cytoplasm through the nuclear pore complex (NPC; 50Mehlin H Daneholt B The Balbiani ring particle a model for the assembly and export of RNPs from the nucleus?.Trends Cell Biol. 1993; 3: 443-447Abstract Full Text PDF PubMed Scopus (32) Google Scholar, 80Visa N Alzhanova-Ericsson A.T Sun X Kiseleva E Björkroth B Wurtz T Daneholt B A pre-mRNA-binding protein accompanies the RNA from the gene through the nuclear pores and into polysomes.Cell. 1996; 84: 253-264Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Importantly, the export of hnRNP A1 to the cytoplasm is specified by a nuclear export signal (NES) sequence, termed M9, that is found on the protein and is devoid of RNA-binding activity (52Michael W.M Choi M Dreyfuss G A nuclear export signal in hnRNP A1 a signal-mediated, temperature-dependent nuclear protein export pathway.Cell. 1995; 83: 415-422Abstract Full Text PDF PubMed Scopus (460) Google Scholar53Michael, W.M., Siomi, H., Choi, M., Piñol-Roma, S., Nakielny, S., Liu, Q., and Dreyfuss, G. (1995b). Signal sequences that target nuclear import and nuclear export of pre-mRNA-binding proteins. In Protein Kinesis. Cold Spring Harbor Symp. Quant. Biol. 60, 663–668.Google Scholar). M9, a 38 amino acid segment near the carboxyl terminus of the protein, is a transferable bifunctional nuclear transport element that can confer both nuclear import (NLS) and nuclear export activity onto heterologous proteins that do not otherwise show these activities. Thus, M9 accesses a specific, signal-mediated, and energy-dependent nuclear cycling pathway that mediates perpetual trafficking of proteins between the nucleus and the cytoplasm. Several hnRNP proteins contain a sequence very similar to M9 (e.g., A2 and B1), and other shuttling hnRNP proteins are likely to have additional NESs. One such novel NES, which bears no sequence similarity to M9, has now been found in the shuttling hnRNP K protein (Dreyfuss). The NESs of the shuttling hnRNP proteins are likely to provide the link to the mRNA nuclear export machinery. The identification of the proteins with which these NESs interact is therefore an important step toward understanding the mechanism of nuclear mRNA export. A candidate M9-binding protein has been found that fulfills the criterion of specific interaction with wild-type M9 but not with transport-defective M9 mutants (Dreyfuss). Further characterization of this protein is now in progress. To ensure against the formation of aberrant proteins, mRNAs that are not fully processed must not be transported to the cytoplasm. This is apparently accomplished by factors bound to unspliced mRNAs that need to be removed before the mRNA can be exported. Spliceosomal snRNPs, and possibly other factors, are likely to provide this retention via the recognition of splice sites, and their subsequent removal from the pre-mRNA then relieves the nuclear restriction as a part of the process of mRNA formation (8Chang D.D Sharp P.A Regulation by HIV Rev depends upon recognition of splice sites.Cell. 1989; 59: 789-795Abstract Full Text PDF PubMed Scopus (388) Google Scholar, 42Legrain P Rosbash M Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm.Cell. 1989; 57: 573-583Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 31Izaurralde E Mattaj I.W RNA export.Cell. 1995; 81: 153-159Abstract Full Text PDF PubMed Scopus (208) Google Scholar). Lentiviruses, including HIV1 and visna, and the oncovirus HTLV-1, have a mechanism designed to bypass this restriction that they utilize to transport to the cytoplasm several of their late-phase unspliced mRNAs. In their unspliced form these pre-mRNAs encode additional proteins, different from those encoded by the corresponding spliced mRNAs, thus ingeniously expanding the repertoire of proteins that can be produced from a small viral genome. This strategy, best characterized in HIV-1, employs the virus-encoded Rev protein which binds to and multimerizes on the viral pre-mRNAs that contain a specific RNA sequence target, the Rev response element (RRE), (10Cullen B.R Malim M.H The HIV-1 Rev protein prototype of a novel class of eukaryotic posttranscriptional regulators.Trends Biochem. Sci. 1991; 16: 346-350Abstract Full Text PDF PubMed Scopus (83) Google Scholar). Rev has two distinct functional domains: the amino-terminal 66 amino acids contain the RRE-binding arginine-rich RNA-binding domain, the NLS, and confer multimerization of Rev on RRE-containing RNA targets; and an activation domain which is comprised of a leucine-rich amino acid sequence (amino acids 73–83 in the 116 amino acid HIV-1 Rev) that is critical for delivering Rev with its bound target RNA to the cytoplasm (11Daly T.J Cook K.S Gray G.S Maione T.E Rusche J.R Specific binding of HIV-1 recombinant Rev protein to the Rev-responsive element in vitro.Nature. 1989; 342: 816-819Crossref PubMed Scopus (264) Google Scholar, 86Zapp M.L Green M.R Sequence-specific RNA binding by the HIV-1 Rev protein.Nature. 1989; 342: 714-716Crossref PubMed Scopus (279) Google Scholar, 9Cullen B.R Mechanism of action of regulatory proteins encoded by complex retroviruses.Microbiol. Rev. 1992; 56: 375-394Crossref PubMed Google Scholar). In definitive recent experiments, the Rev activation domain has been shown to function as a potent NES (16Fischer U Huber J Boelens W.C Mattaj I.W Lührmann R The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs.Cell. 1995; 82: 475-483Abstract Full Text PDF PubMed Scopus (965) Google Scholar, 82Wen W Meinkoth J.L Tsien R.Y Taylor S.S Identification of a signal for rapid export of proteins from the nucleus.Cell. 1995; 82: 463-473Abstract Full Text PDF PubMed Scopus (972) Google Scholar, 51Meyer B.E Meinkoth J.L Malim M.H Nuclear transport of human immunodeficiency virus type 1, visna virus, and equine infectious anemia virus Rev proteins identification of a family of transferable nuclear export signals.J. Virol. 1996; 70: 2350-2359Crossref PubMed Google Scholar) and to mediate the binding of Rev to a cellular protein termed Rab (6Bogerd H.P Fridell R.A Madore S Cullen B.R Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins.Cell. 1995; 82: 485-494Abstract Full Text PDF PubMed Scopus (278) Google Scholar) or hRIP (19Fritz C.C Zapp M.L Green M.R A human nucleoporin-like protein that specifically interacts with HIV Rev.Nature. 1995; 376: 530-533Crossref PubMed Scopus (230) Google Scholar). Rab/hRIP is a 60 kDa protein that contains numerous FG dipeptide repeats characteristic of nucleoporins, suggesting that it functions directly in the transport of proteins across the NPC (12Davis L.I The nuclear pore complex.Annu. Rev. Biochem. 1995; 64: 865-896Crossref PubMed Scopus (283) Google Scholar). The Rev NES is highly homologous to a leucine-rich NES found in the protein kinase inhibitor (PKI; 82Wen W Meinkoth J.L Tsien R.Y Taylor S.S Identification of a signal for rapid export of proteins from the nucleus.Cell. 1995; 82: 463-473Abstract Full Text PDF PubMed Scopus (972) Google Scholar) and in the 5S rRNA-binding protein TFIIIA. Cullen and colleagues (HHMI, Duke University, Durham, North Carolina) reported that the PKI and TFIIIA NESs can functionally substitute for the Rev NES and that the PKI NES can also bind directly to Rab/hRIP. Using a randomization–selection approach in the yeast two-hybrid system, they have further defined the critical residues of the leucine-rich Rex NES and found a remarkably relaxed consensus: L-X2-3-L/I/V/F-X2-3-L-X-L/I. It appears that X can be almost any amino acid except proline. TFIIIA can serve to transport 5S rRNA, at least in oocytes (26Guddat U Bakken A.H Pieler T Protein-mediated nuclear export of RNA 5S rRNA containing small RNPs in Xenopus oocytes.Cell. 1990; 60: 619-628Abstract Full Text PDF PubMed Scopus (166) Google Scholar), and Rev NES coupled to bovine serum albumin competes with 5S rRNA nuclear export in Xenopus oocytes but does not compete with mRNA export. It is therefore likely that the lentiviruses' strategy for overcoming the nuclear retention of unspliced pre-mRNAs is to bypass splicing of their RRE-containing pre-mRNAs by transporting them to the cytoplasm via a nuclear export pathway that the cell does not normally use for nuclear export of mRNAs and which may not be subject to the surveillance system that recognizes introns. M. Rosbash (HHMI, Brandeis University, Waltham, Massachusetts) and coworkers have recently shown that, remarkably, Rev also functions in yeast, albeit at lower efficiency than in higher eukaryotes (74Stutz F Rosbash M A functional interaction between Rev and yeast pre-mRNA is related to splicing complex formation.EMBO J. 1994; 13: 4096-4104Crossref PubMed Scopus (68) Google Scholar). They have now shown that the RRE RNA structure that forms in yeast is similar to that determined from in vitro studies. Wild-type Rev, but not Rev activation domain transport–defective mutants, interacts with a yeast protein termed RIP1 (75Stutz F Neville M Rosbash M Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast.Cell. 1995; 82: 495-506Abstract Full Text PDF PubMed Scopus (202) Google Scholar). 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• W2000371296 title "From Transcript to Protein" @default.
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