From Transcript to Protein Meeting Review

Cell, Vol. 85, 963–972, June 28, 1996, Copyright 1996 by Cell Press From Transcript to Protein Meeting Review

Gideon Dreyfuss,* Matthias Hentze,† additional protein splicing factors. Recognition of in- and Angus I. Lamond‡ trons involves both protein–RNA and RNA–RNA interac- *Howard Hughes Medical Institute tions being made by splicing factors with conserved Department of Biochemistry and Biophysics sequence elements on the pre-mRNA at the 5Ј and 3Ј University of Pennsylvania School of Medicine intron–exon junctions and at the branch site (reviewed Philadelphia, Pennsylvania 19104-6148 by Moore et al., 1993; Nilsen, 1994). Considerable effort † European Molecular Biology Laboratory is currently being directed toward identifying the protein Meyerhofstrasse 1 components of spliceosomes, determining how they in- Postfach 102209 teract with pre-mRNA and with each other during the D69117 Heidelberg splicing reaction, and analyzing how these interactions Germany are regulated. ‡ Department of Biochemistry B. Seraphin (EMBL, Heidelberg) and P. Legrain (Insti- University of Dundee tut Pasteur, Paris) reported studies on how 5Ј splice Dundee DD1 4HN sites and branch sites in budding yeast pre-mRNAs are United Kingdom recognized by splicing factors. Legrain described an exhaustive mutagenesis of the branch point consensus sequence, analyzed using an in vivo assay system, One of the most interesting formats for a scientific meet- which showed that certain base substitutions could dif- ing involves bringing together researchers working in ferentially affect the splicing of pre-mRNA and its trans- overlapping or closely related fields where the opportu- port out of the nucleus. Although base pairing at the nity can arise for cross-fertilization and exchange of branch point between the pre-mRNA and U2 snRNA ideas. This was the aim of the recent workshop on mRNA is essential for splicing, it is apparently not linked to processing, transport, and translation held at the Juan retention of unspliced pre-mRNA in the nucleus. Legrain March Foundation in Madrid on March 11–13. All the reported that mutational data also point to an important fields represented shared a common interest in studying RNA–protein interaction taking place at the nucleotide the synthesis, maturation, transport, stability, and func- immediately upstream of the branch site consensus, tion of mRNA. The presentations included talks on char- where a U residue is moderately conserved. B. Seraphin acterizing the machineries involved in splicing, polyade- (EMBL, Heidelberg) described how the mechanism for nylating, and translating mRNA; how RNAs and proteins selecting the precise site of cleavage at the 5Ј intron– are transported into and out of the nucleus; how post- exon junction involves a complex network of snRNA– translational mechanisms can control gene expression; pre-mRNA base pairing. First, the region to be cleaved and how RNA mechanisms are involved in virus–host is selected by base pairing between the 5Ј terminus of interactions. The venue was ideal, our hosts were gener- U1 snRNA and the pre-mRNA. Subsequently, both U5 ous and helpful, and the outcome was a tremendously and U6 snRNAs participate in selecting the exact bond stimulating meeting where every talk was followed by to be cleaved by base pairing to the pre-mRNA. Seraphin an in-depth, open discussion, usually lasting 10–15 mi- pointed out that there may be some redundancy in this nutes. Many of these discussions resumed in the eve- mechanism, as certain point mutations at the 5Ј splice nings over Tapas and, as it is the Spanish custom to site that alter splice site choice can be suppressed by dine late, continued into the small hours. We hope that introducing mutations into either U5 or U6 snRNAs that the Juan March Foundation will decide to stage a repeat affect their potential base pairing interactions with the performance in the future. pre-mRNA. Splicing snRNPs are associated with a common com- Spliceosomes, Splicing Factors, plex of eight proteins, called Sm proteins, that bind and Polyadenylation directly to U1, U2, U4, and U5 snRNAs but not to U6. A Spliceosomesare the large RNA–protein complexes that conserved “Sm domain” has been identified atthe amino catalyze excision of introns from mRNA precursors (pre- termini of Sm proteins and used to identify new members mRNA) in the nucleus. Introns are spliced by a two- of the Sm protein family in the sequence database (B. step mechanism, both steps being transesterification Seraphin). In yeast, 15 Sm-type proteins have been iden- reactions (reviewed by Moore et al., 1993). First, cleav- tified and several of these have been shown to associate age occurs at the 5Ј intron–exon junction, generating a with snRNAs in vivo. Interestingly, most of the Sm-type free 5Ј exon and a branched intron–3Ј exon as reaction proteins can be subdivided into two groups that appear intermediates. The branched structure is formed by the to form separate complexes. One complex corresponds esterification of the 5Ј end of the intron to a 2Ј hydroxyl to the previously known set of Sm proteins that bind to group of an adenosine residue (branch site), close to U1, U2, U4, and U5 snRNAs, while the other corresponds the 3Ј end of the intron. The second step produces to a new complex that binds to U6 snRNA (B. Seraphin). ligated exons (mRNA) and a fully excised branched in- Another advance facilitated by the burgeoning se- tron as the reaction products. The major subunits of quence databases is the isolation of a human homolog spliceosomes are the U1, U2, U4/U6, and U5 small nu- of the yeast splicing factor prp18, which was identified clear ribonucleoprotein particles (snRNPs), which as- and cloned using information derived from nematode semble on pre-mRNAs in a stepwise pathway along with and rice EST sequences (A. Krainer, Cold Spring Harbor). Cell 964

Like its yeast counterpart, human prp18 protein is re- is whether other minor snRNAs are also in the ATAC quired for the second catalytic step of the splicing re- spliceosomes and playing the role of U4/U6 snRNP. It action. Either the yeast or human prp18 proteins will is also unclear just how many protein splicing factors complement a HeLa splicing extract that has been im- will be shared by the two forms of spliceosome. Spliceo- munodepleted of endogenous prp18, although the hu- somes purified from extracts containing two distinct man gene will not complement a yeast prp18 mutant in forms of U1 snRNA, which differ in the sequence of their vivo (A. Krainer). A conserved family of protein splicing loop B regions, were reported to contain disproportion- factors found in Drosophila and human cells share a ately more of the U1b variant (M. Bach, Barcelona, dipeptide repeat motif consisting of alternating serine Spain). It therefore appears that spliceosome structure and arginine residues (reviewed by Fu, 1995). These may be more heterogeneous than was previously “SR” proteins are involved in both constitutive and alter- thought. A. Lamond (University of Dundee, Scotland) native splicing mechanisms and the family can be subdi- reported a collaborative project by the groups of La- vided into two groups, typified respectively by the pro- mond and Mann to systematically identify all the proteins SF2/ASF and SC35 (A. Krainer). These two groups teins in the mammalian spliceosome. This project takes of proteins differ in their substrate specificity in constitu- advantage of the powerful new developments in native splicing and in their mode of activating alternative noelectrospray mass spectrometry (Wilm et al., 1996), splicing. Krainer reported that the analysis of chimeric which facilitate mass and sequence analysis of purified proteins, formed by swapping domains from SF2/ASF proteins in levels as low as 0.1 pmol. In combination and SC35, has shown that the substrate specificities with the rapidly expanding database of EST clones, it of the proteins correlate with the presence of specific is hoped that this approach will accelerate the charac- constituent domains, particularly the RNP motif RNA- terization of new splicing factors and make the clones binding domains (RNA recognition motif). However, be- available for future detailed functional studies (A. cause different SR protein modules appear to be impor- Lamond). tant with different substrates, Krainer noted that this By far the most lively discussion at the meeting was analysis has not so far uncovered any simple rules for stimulated by the astonishing presentation from C. predicting the substrate specificity of, or the alternative Milstein (MRC, Cambridge, England), who addressed splicing patterns mediated by, a particular SR protein. the mechanism whereby B cells eliminate mRNAs con- J. Valcarcel (EMBL, Heidelberg) presented new data on taining premature stop codons. These stop codons arise the function of U2AF, an essential, conserved splicing as a result of the DNA rearrangements that occur when factor that also has SR dipeptide repeats but differs in B cells mature in response to antigen. Ig␬ light chain its properties from SR proteins such as ASF/SF2 and mRNAs containing premature stop codons are ex- SC-35. U2AF has two subunits, both containing SR repressed at very low levels (Lozano et al., 1994). The peat motifs, and binds directly to the polypyrimidine mechanism responsible for this effect appears to take tract of pre-mRNAs through RNA binding domains in place in the nucleus and involve an inhibition of splicing the large subunit, which do not require the SR motif to of ␬ light chain pre-mRNAs containing premature stop bind RNA. However, the SR repeats in the large subunit codons (Aoufouchi et al., 1996). Milstein reported that are required to stabilize the binding of U2 snRNP at the this effect is both cell type– and gene-specific and could branch point and mutagenesis shows that (SR)7 is the be reproduced in a soluble S100 system, which would minimum functional unit of the motif. Valcarcel reported appear to exclude the involvement of intact ribosomes that the arginine can be substituted by lysine but not or feedback from the translation system to the spliceo- by alanine, while the serine can be changed to glycine, some. How do B cells screen pre-mRNAs in the nucleus methionine, or threonine without loss of function but for in frame stop codons? Answering this question is cannot be replaced by negatively charged amino acids. clearly going to be a major focus of future work. The run of positive charges, which directly contacts the W. Keller (Biozentrum, Basel, Switzerland) reported pre-mRNA at the branch site, may stabilize base pairing on progress in studying the mechanism of polyadenyla- between the branch site and U2 snRNA. tion of nuclear pre-mRNA. This involves a two-step A particularly exciting new development was the re- mechanism in which the substrate is first cleaved down- port from J. Steitz (HHMI, Yale Medical School, New stream of the conserved AAUAAA motif, then a tail of Haven) showing that a novel form of mammalian spliceo- 150–250 adenosine residues is polymerized onto the some containing U5 snRNP and the minor snRNPs U11 free 3Ј terminus. Addition of the poly(A) tail involves an and U12, but not U1, U2, or U4/U6, assemble in vitro initial oligoadenylation at the 3Ј end of the cleaved on a minor class of introns (called AT-AC introns) with mRNA followed by binding of a protein called PABII and noncanonical consensus splice sites (Tarn and Steitz, subsequent extension of the tail (reviewed by Keller, 1996). This confirms an earlier prediction by Hall and 1995; Wahle and Keller, 1996). Considerable advances Padgett that U11 and U12 snRNPs may substitute for have been made in cloning and characterizing the com- U1 and U2 snRNPs during splicing of AT-AC introns ponents of the polyadenylation machineries from both (Hall and Padgett, 1994). Recent studies by the same mammals and yeastand numerous homologs have been authors have also shown that U12 snRNA base pairs identified in the two systems (W. Keller). The polyadenyl- with the branch point of AT-AC introns in vivo (Hall and ation machinery is large and surprisingly complex, in- Padgett, 1996). Steitz reported that U5 snRNP function cluding factors specifically involved in either cleavage is required for splicing both classes of introns, while U1 or poly(A) addition and factors required for both steps. and U2 snRNPs are not required for splicing AT-AC CPSF is a mammalian factor required for both steps introns. Conversely, U11 and U12 appear only involved that has a subunit of 160 kDa which binds directly to in splicing ATAC introns. A major question that remains the AAUAAA motif. CPSF and poly(A) polymerase (PAP) Meeting Review 965

Figure 1. Gems Are a Novel Structure Re- lated to Coiled Bodies Superimposed laser confocal microscopy images of indirect double-label immunofluorescence of HeLa cells using anti-SMN antibody 2B1 (green) and anti-p80 coilin antibody P␦ (red) are shown on the left. The colocalization of red and green labeling results in a yellow color. The corresponding differential interfer- ence contrast images are shown on the right. Gems are indicated by arrows. For further details, see Liu and Dreyfuss, 1996.

are sufficient in vitro to polyadenylate a precleaved sub- interactions with trans-acting factors including spliceo- strate containing the AAUAAA motif. Keller reported that somal snRNPs, and remain associated with the pro- the catalytic core of PAP lies at the amino terminus and cessed nuclear mRNAs (reviewed by Dreyfuss et al. contains at least three essential aspartate residues that, 1993). Several observations suggest that hnRNP pro- if mutated, severely reduce Kcat for polyadenylation. The teins also have a role in the export of mRNAs from the catalytic domain of PAP shows similarity to the catalytic nucleus to the cytoplasm. First, several of the abundant domains of other nucleotidyltransferases, notably tRNA hnRNP proteins, including A1, A2, D, I, and K, shuttle CCA adding enzyme, DNA terminal transferase and DNA continuously and rapidly between the nucleus and the polymerase ␤ (Martin and Keller, 1996). cytoplasm (Pin˜ ol-Roma and Dreyfuss, 1992, 1993). Sec- The nucleus is a highly structured organelle with many ond, the shuttling hnRNP proteins, specifically A1, are nuclear antigens concentrated in specific subnuclear associated with mRNA both in the nucleus and in the domains. The groups of A. Lamond and G. Dreyfuss cytoplasm (Pin˜ ol-Roma and Dreyfuss, 1992, 1993); their (HHMI, University of Pennsylvania, Philadelphia) pre- association with cytoplasmic mRNAs is brief, as they sented new studies on distinct classes of subnuclear normally return to the nucleus rapidly. Third, an A1 ho- domains called, respectively, coiled bodies and gems molog has been observed to be associated with a spe- (see Figure 1). Coiled bodies contain splicing snRNPs, cific mRNA, the Balbiani ring mRNA, as it is being trans- but not the SR family of protein splicing factors, and are located to the cytoplasm through the nuclear pore found either closely associated with the periphery of complex (NPC; Mehlin and Daneholt, 1993; Visa et al., the nucleolus or free in the nucleoplasm (reviewed by 1996). Importantly, the export of hnRNP A1 to the cyto- Bohmann et al., 1995). Lamond reported a combination plasm is specified by a nuclear export signal (NES) se- of mutational data and studies with phosphatase inhibi- quence, termed M9, that is found on the protein and is tors showing that coiled bodies can be induced to form devoid of RNA-binding activity (Michael et al., 1995a, within nucleoli and suggested that trafficking of coiled 1995b). M9, a 38 amino acid segment near the carboxyl bodies between the nucleolus and nucleoplasm may be terminus of the protein, is a transferable bifunctional controlled by a protein phosphorylation mechanism. A nuclear transport element that can confer both nuclear new class of subnuclear body has been identified that import (NLS) and nuclear export activity onto heterolo- contains the protein encoded by the survival of motor gous proteins that do not otherwise show these activi- neuron (SMN) gene (G. Dreyfuss). This has been identi- ties. Thus, M9 accesses a specific, signal-mediated, and fied as the determining gene for spinal muscular atrophy energy-dependent nuclear cycling pathway that medi- (SMA), a fatal autosomal recessive disease causing pro- ates perpetual trafficking of proteins between the nu- gressive muscular wasting and paralysis (Lefebvre et cleus and the cytoplasm. Several hnRNP proteins con- al., 1995). Dreyfuss described how the SMN protein is tain a sequence very similar to M9 (e.g., A2 and B1), specifically localized in subnuclear bodies that are simi- and other shuttling hnRNP proteins are likely to have lar to coiled bodies in size and number, but are distinct additional NESs. One such novel NES, which bears no structures that do not contain splicing snRNPs. They sequence similarity to M9, has now been found in the have been called gems, for Gemini of coiled bodies (Liu shuttling hnRNP K protein (Dreyfuss). The NESs of the and Dreyfuss, 1996). Future studies will be aimed at shuttling hnRNP proteins are likely to provide the link discovering the precise biolgical functions of coiled to the mRNA nuclear export machinery. The identifica- bodies, gems, and other classes of subnuclear bodies tion of the proteins with which these NESs interact is in which specific nuclear antigens concentrate. therefore an important step toward understanding the mechanism of nuclear mRNA export. A candidate M9- Nuclear Export of mRNAs and binding protein has been found that fulfills the criterion Spliceosomal snRNAs of specific interaction with wild-type M9 but not with Heterogeneous nuclear ribonucleoprotein particle transport-defective M9 mutants (Dreyfuss). Further (hnRNP) proteins are abundant pre-/mRNA-binding pro- characterization of this protein is now in progress. teins that bind to nascent transcripts, influence their To ensure against the formation of aberrant proteins, Cell 966

mRNAs that are not fully processed must not be trans- M. Rosbash (HHMI, Brandeis University, Waltham, ported to the cytoplasm. This is apparently accom- Massachusetts) and coworkers have recently shown plished by factors bound to unspliced mRNAs that need that, remarkably, Rev also functions in yeast, albeit at to be removed before the mRNA can be exported. lower efficiency than in higher eukaryotes (Stutz and Spliceosomal snRNPs, and possibly other factors, are Rosbash, 1994). They have now shown that the RRE likely to provide this retention via the recognition of RNA structure that forms in yeast is similar to that deter- splice sites, and their subsequent removal from the pre- mined from in vitro studies. Wild-type Rev, but not Rev mRNA then relieves the nuclear restriction as a part of activation domain transport–defective mutants, inter- the process of mRNA formation (Chang and Sharp, 1989; acts with a yeast protein termed RIP1 (Stutz et al., 1995). Legrain and Rosbash, 1989; Izaurralde and Mattaj, RIP1 is a novel yeast protein which is a member of the 1995). Lentiviruses, including HIV1 and visna, and the FG repeat–bearing nucleoporin family and is thus likely oncovirus HTLV-1, have a mechanism designed to by- to be a functional homolog of the mammalian Rab/RIP. pass this restriction that they utilize to transport to the In a recent, more extensive two-hybrid investigation of cytoplasm several of their late-phase unspliced mRNAs. the nucleoporin family, Rosbash’s laboratory has identi- In their unspliced form these pre-mRNAs encode addi- fied additional yeast and mammalian nucleoporins that tional proteins, different from those encoded by the cor- interact with Rev as strongly as RIP1 or mammalian responding spliced mRNAs, thus ingeniously expanding Rab/RIP. Analysis of deletion constructs indicates that the repertoire of proteins that can be produced from a it is the nucleoporin FG and GLFG repeat regions that small viral genome. This strategy, best characterized in interact with Rev. These results further suggest that Rev HIV-1, employs the virus-encoded Rev protein which directly promotes the nuclear export of RRE-containing binds to and multimerizes on the viral pre-mRNAs that transcripts by targeting them to the NPC. contain a specific RNA sequence target, the Rev re- Using genetic enrichment and screening procedures sponse element (RRE), (Cullen and Malim, 1991). Rev in yeast, many conditional temperature-sensitive mRNA has two distinct functional domains: the amino-terminal transport mutants, which accumulate poly(A)-contain- 66 amino acids contain the RRE-binding arginine-rich ing RNA in the nucleus, have been obtained (reviewed RNA-binding domain, the NLS, and confer multimeriza- in Schneiter et al., 1995; see also, Amberg et al., 1992; tion of Rev on RRE-containing RNA targets; and an acti- Kadowaki et al., 1992). Several of the corresponding vation domain which is comprised of a leucine-rich genes have been cloned and their protein products have amino acid sequence (amino acids 73–83 in the 116 been characterized. A. Tartakoff (Case Western Reserve amino acid HIV-1 Rev) that is critical for delivering Rev University) described three such mutants (termed MTRs, with its bound target RNA to the cytoplasm (Daly et al., for mRNA transport mutants), MTR13, MTR4, and MTR1. 1989; Zapp and Green, 1989; Cullen, 1992). In definitive MTR13 encodes an RNA-binding protein that has some recent experiments, the Rev activation domain has been overall sequence similarity to hnRNPs A1 and A2, includ- shown to function as a potent NES (Fischer et al., 1995; ing two amino-terminal RNP motifs and a glycine-rich Wen et al., 1995; Meyer et al., 1996) and to mediate the carboxy-terminal domain, and, consistent with this, it binding of Rev to a cellular protein termed Rab (Bogerd has previously been shown to be a nuclear poly(A)ϩ et al., 1995) or hRIP (Fritz et al., 1995). Rab/hRIP is a RNA-binding (hnRNP) protein called NOP3/NPL3/NAB1 60 kDa protein that contains numerous FG dipeptide (Russell and Tollervey, 1992; Flach et al., 1994; Wilson repeats characteristic of nucleoporins, suggesting that et al., 1994). Interestingly, Mtr13p, like A1 (Pin˜ ol-Roma it functions directly in the transport of proteins across and Dreyfuss, 1991), shuttles between the nucleus and the NPC (Davis, 1995). The Rev NES is highly homolo- the cytoplasm and accumulates in the cytoplasm after gous to a leucine-rich NES found in the protein kinase RNA polymerase II inhibition. Mtr4p is a 100 kDa protein inhibitor (PKI; Wen et al., 1995) and in the 5S rRNA- that contains DEAD box motifs, a characteristic of RNA binding protein TFIIIA. Cullen and colleagues (HHMI, helicases, as well as five repeats of a leucine-rich motif Duke University, Durham, North Carolina) reported that reminiscent of the HIV/PKI/TFIIIA NESs. The functional the PKI and TFIIIA NESs can functionally substitute for significance of these sequences in Mtr4p are, however, the Rev NES and that the PKI NES can also bind directly not yet known. It is intriguing that, at the nonpermissive to Rab/hRIP. Using a randomization–selection approach temperature, mtr4 temperature-sensitive mutants accu- in the yeast two-hybrid system, they have further defined mulate poly(A)ϩ RNA in the yeast nucleolus (Tartakoff). the critical residues of the leucine-rich Rex NES and This, together with similar observations for some other found a remarkably relaxed consensus: L-X2-3-L/I/V/F- yeast mRNA export mutants, suggests that a role for X2-3-L-X-L/I. It appears that X can be almost any amino the nucleolus as a way station in mRNA export from the acid except proline. TFIIIA can serve to transport 5S nucleus needs to be considered (Schneiter et al., 1995; rRNA, at least in oocytes (Guddat et al., 1990), and Rev Tani et al., 1995). MTR1 (also known as PRP20 or SRM1) NES coupled to bovine serum albumin competes with encodes the Saccharomyces cerevisiae homolog of 5S rRNA nuclear export in Xenopus oocytes but does RCC1, a mammalian guanine nucleotide exchange fac- not compete with mRNA export. It is therefore likely tor that regulates the GTPase, Ran/TC4 (Sazer, 1996). that the lentiviruses’ strategy for overcoming the nuclear A role for the Ran/TC4 GTPase cycle in nuclear protein retention of unspliced pre-mRNAs is to bypass splicing import has been clearly demonstrated (Melchior et al., of their RRE-containing pre-mRNAs by transporting 1993; Moore and Blobel, 1993; Sweet and Gerace, 1995), them to the cytoplasm via a nuclear export pathway that and the finding that a GTPase cycle is likely to be re- the cell does not normally use for nuclear export of quired for RNA export further highlights the requirement mRNAsand which may not be subject to the surveillance for shared components for both nuclear import and ex- system that recognizes introns. port. Among the additional components now apparently Meeting Review 967

required for both transport processes are components conserving the domains in Staufen protein described of the NPC, several of which have been found to be as responsible for bicoid mRNA binding. The widely involved in transport using biochemical and genetic ap- expressed hSTL protein showed a cytosolic pattern of proaches (Bogerd et al., 1994; Wente and Blobel, 1994; immunofluorescence staining; however, its coexpres- Gorsch et al., 1995; Grandi et al., 1995). For most of the sion with NS1 leads to drastic relocalization and it accu- yeast genes implicated in RNA export, disruption leads mulates in nuclear foci that also strictly localize with the to phenotypes in addition to that of accumulated po- NS1 protein. In addition, hSTL protein could be coimmu- ly(A)ϩ RNA in the nucleus. Although the pleiotropic na- noprecipitated with NS1 from cells coexpressing both ture of the mutations makes it unclear whether these proteins. proteins have a primary role in RNA transport, the yeast Two major themes thus emerge from recent work on genetic approach has potential to yield insight into the RNA transport. First, the RNA-binding proteins that are mechanism of RNA export. specifically associated with the RNAs play a cardinal An exciting example of the cell’s innovative use of the role in determining the localization and transport of the same components for different pathways comes from RNAs. In turn, the sequence of the RNA itself, as well work from the Mattaj, Go¨ rlich, and Laskey laboratories as the polymerase complex that forms it—and thus the on the nuclear export of U snRNAs (E. Izaurralde, EMBL, specific promoters—determines the RNP proteins that Heidelberg). U snRNAs (except for U6) are transcribed assemble on it. Second, many of the machineries that by RNA polymerase II and acquire an m7G cap, to which transport RNAs, both the soluble factors and the under- the cap binding complex (CBC) binds. The CBC is com- lying cellular structures (e.g., the NPC), are also involved prised of two subunits, CBP80, which contains a classi- in the transport of proteins both into and out of the cal bipartite NLS, and CBP20, which contains a single nucleus. A reliable in vitro system for studying nuclear RNP motif RNA-binding domain (Ohno et al., 1990; Ka- export of RNAs has not been developed, but in vivo taoka et al., 1994). The CBC, which also binds to the approaches, including injections into amphibian oo- caps of pre-mRNAs and is important in their splicing cytes and genetic analyses in yeast and viral systems, (Izaurralde et al., 1994), has been shown to be an essen- promise to continue to yield important and exciting infor- tial export factor for U snRNAs (Izaurralde et al., 1995). mation on RNA transport. Much will certainly be learned In an elegant series of experiments, these workers have from characterization of the proteins that interact with now shown that the NLS receptor subunits, importin ␣ the NESs on the shuttling hnRNP proteins and other and ␤, mediate a cycle of association, dissociation, and RNA-binding proteins. The RNA-binding proteins, which transport of the CBC and its associated U snRNAs. Ac- play key roles in every aspect of the metabolism of cording to this model, the importin ␣–CBC complex RNAs, have distinct domains for RNA binding, and the binds to capped snRNAs in the nucleus and accompan- structures of several of these RNA-binding domains ies them to the cytoplasm. In the cytoplasm, importin ␤ have already been determined (Burd and Dreyfuss, binds to importin ␣ and this triggers the release of the 1994; Nagai,1996). In addition to the structural methods, snRNA from the complex. Importins ␣ and ␤ then medi- considerable new insight into the interactions of RNA- ate the import of the CBC to the nucleus, where importin binding proteins with RNA will likely emerge from appli- ␣ remains bound to it to initiate a new transport cycle. cation of recently developed genetic approaches. J. Be- Importin ␤, which does not accumulate in the nucleus, lasco (Harvard Medical School) described the use of separates from the complex and is rapidly returned to two such systems: phage display and selection/screen- the cytoplasm to effect the release of another round of ing by repression of translation in E. coli. In the phage exported snRNAs. Though further direct evidence for display scheme, a mutagenized stockof an RNA-binding the involvement of importins in snRNA export is needed, domain (the U1 snRNA-binding U1A first RNP motif was this mechanism of release of exported RNAs is likely to used) is displayed on the surface of a phage, and mu- be an important general feature of the transport cycle tants of a desired characteristic, such as changed affin- of many RNAs. ity or specificity, are selected (Laird-Offringa and Be- As exemplified by HIV, discussed here, studies of viral lasco, 1995). In the second scheme, a mutagenized gene expression can provide invaluable insight into cel- stock of an RNA-binding protein (HIV1 Rev was used) lular RNA processing and transport mechanisms. The is expressed in E. coli, which also expresses the lacZ influenza virus encodes several proteins that mediate gene in whose mRNA the binding site for the RNA-bind- production of viral particles by regulating both viral and ing protein is placed just upstream of the Shine– cellular transcriptprocessing. Forexample, the NS1 pro- Delgarno box. Blue colonies will appear whenever a tein mediates a generalized retention of polyadenylated mutant RNA-binding protein that fails to bind to lacZ RNA in the nucleus as well as a modulation in the splicing mRNA is expressed, as a result of its failure to repress of pre-mRNAs (Fortes et al., 1994; Lu et al., 1994). In translation of the lacZ gene. Both schemes, and addi- addition, it enhances the rate of translation initiation of tional ones that can be devised, promise to increase viral, but not cellular, mRNAs (Enami et al., 1994). This our knowledge of how RNA-binding proteins interact plethora of effects has been linked to the NS1 protein with and affect their RNA targets. (Enami et al., 1994; Qiu et al., 1995). J. Ortin and colleagues (Centro de Biologia Molecular, CSIC, Madrid) have used the two-hybrid genetic trap to screen a human Cytoplasmic mRNPs: Cap, Poly(A), cDNA fusion library using NS1 protein as a bait. An and End-to-End Communication interacting clone, hSTL (human STaufen-Like), showed The 5Ј and 3Ј untranslated regions (UTRs) of mRNAs can a 55% similarity to the D. melanogaster Staufen protein, contain cis-acting elements that control the translation, Cell 968

share a short motif of amino acid homology that medi-

ates their binding to eIF-4E (T50RIIYDRKFLMEC62 in 4E-

BP1 and E412KKRYDREFLLGF424 in eIF-4G [identical amino acids are underlined]). When 4E-BP1 binds to eIF-4E, the eIF-4G interaction site—and thus eIF-4F assembly—is competitively blocked (Haghighat et al., 1995). This function of 4E-BPs is regulated by metabolic signaling. Insulin and growth factors can induce the

phosphorylation of serine65 in 4E-BP1, which is conserved in 4E-BP2 and located close to the eIF-4E interaction site, resulting in abolished binding to eIF-4E (Lin et al., 1994; Pause et al., 1994). The signal transduction Figure 2. End-to-End Communication in Cytoplasmic mRNA Me- pathway that leads to 4E-BP phosphorylation is rapam- tabolism ycin- and wortmannin-sensitive (Beretta et al., 1996) and A capped, polyadenylated mRNA is depicted to be subject to inter- thus distinct from the MAP kinase pathway implicated actions and modifications that stimulate (green) or inhibit (red) its earlier. expression in the cytoplasm. CPEB, cytoplasmic polyadenylation Not all mRNAs initiate translation by a cap-dependent element binding protein; dcp1, Saccharomyces cerevisiae decap- mechanism that is sensitive to 4E-BPs. E. Martinez- ping protein 1; IRP, ironregulatory protein; 4E-BPs, eukaryotic trans- Salas (Universidad Autonoma de Madrid, Spain) and lation initiation factor 4E-binding protein; m, methylation of 2Ј-O- ribose of the first and second transcribed nucleotide. G. Belsham (Institute for Animal Health, Pirbright, UK) reported studies on picornaviral RNAs which are translated by a cap-independent mechanism that requires a degradation, and localization of transcripts. In addition, highly structured region of several hundred nucleotides the 5Ј and 3Ј terminal nuclear modifications of eukaryotic in the 5Ј UTRs of the viral RNAs, the internal ribosome 7 mRNAs, the cap [ mGpppN] and the poly(A) tail, play entry site (IRES). Martinez-Salas isolated a hypervirulent critical roles for mRNA translation and stability. Work foot-and-mouth disease virus after one hundred pas- from many laboratories during the past years has shown sages of persistent infection, whose IRES activity is en- that the binding of a heterotrimeric complex, eIF-4F, to hanced up to 5-fold in comparison to the parental virus. the cap structure is required to initiate the assembly Characterization of this and 15 other mutants under- of a translation-competent ribosome on most cellular scored the importance of the stability of the structured mRNAs. The role of the cap in mRNA stabilization has domain 3 (in their nomenclature, Martinez-Salas et al., been demonstrated most clearly in S. cerevisiae, where 1993) of the IRES, and revealed a minor contribution of decapping of the 5Ј terminus represents part of a com- the primary sequence in this domain to IRES function. mon degradation pathway for stable and unstable Belsham demonstrated that nonfunctional IRES variants mRNAs. Decapping is preceded by deadenylation at the can be complemented efficiently in trans by coexpres- 3Ј terminus, which reflects the role of the poly(A) tail in sion of either an intact IRES, or a second IRES with a mRNA turnover and indicates communication between nonoverlapping defect. This trans-complementation has the two termini (see below). Several presentations at been shown for entero-, cardio-, and aphtoviral IRES in this meeting addressed the involvement of the cap and vivo but has not yet been reconstituted in a cell-free the poly(A) tail in translational regulation and discussed translation system. It requires substantial sequence examples of end-to-end communication in translation identity between the two complementing IRES ele- and mRNA degradation (summarized in Figure 2). After mRNAs have been released from the nuclear ments, suggesting a requirement for RNA–RNA interac- export machinery (Izaurralde), they are bound by a cyto- tions. It might reflect a concentration of mRNAs in plasmic cap-binding complex, eIF-4F, that is composed mRNPs that could serve as sites for IRES-dependent of three subunits: eIF-4E (which directly contacts the translation (Belsham). 7mG cap), the RNA helicase eIF-4A (not depicted in Fig- When mRNAs are translated by a cap-dependent ure 2), and eIF-4G (formerly called p220). eIF-4E is mechanism, the cap-proximal region of the transcript thought to represent a rate-limiting factor in translation serves as the entry site for the small ribosomal subunit. (Sonenberg, 1996), predisposing it as a potential target Proteins binding with high affinity to sequences within for translational control. Its overexpression in mamma- this region block the ribosomal entry step and repress lian cell lines induces changes in cell morphology, trans- translation (Gray and Hentze, 1994; Stripecke et al., formation, focus formation in soft agar, and malignant 1994). An example of this mechanism of translational growth in nude mice (De Benedetti and Rhoads, 1990; control in somatic cells is the regulation of mRNAs en- Lazaris-Karatzas et al., 1990). N. Sonenberg (McGill Uni- coding proteins involved in iron metabolism by iron- versity, Montreal, Canada) summarized the cloning of responsive elements (IREs) and iron regulatory proteins complementary DNAs encoding a family of small pro- (IRPs). M. Hentze (EMBL, Heidelberg) reported the sur- teins that can bind to eIF-4E (4E-BPs) and inhibit its prising finding that IRP-mediated translational repres- function. The overexpression of 4E-BP1 or 4E-BP2 in sion induces partial deadenylation of ferritin L-chain cells transformed by eIF-4E reduces their growth in soft mRNA. The deadenylation appears to represent a con- agar and slows tumor growth in nude mice (Sonenberg). sequence rather than the cause of translational inhibi- The molecular mechanism underlying their inhibition of tion, since ferritin H-chain mRNA, which bears only a eIF-4E function has been defined: 4E-BP1 and eIF-4G short poly(A) tail (30–70 A’s) when being translated, is Meeting Review 969

efficiently repressed by IRPbinding but not further dead- could be a methyltransferase that is complexed with enylated. Moreover, partial deadenylation is also ob- poly(A) polymerase. Interestingly, precedence for com- served with both IRE-regulated reporter mRNAs car- plex formation between the nucleoside-2Ј-O-methyl- rying long poly(A) tails and reporter mRNAs that are transferase and poly(A) polymerase has been reported translationally repressed by other RNA binding proteins for vaccinia virus (Schnierle et al., 1992). that occupy a cap-proximal site. Thus, the IRE repre- Finally, 3Ј-to-5Ј end communication forms the basis of sents a polyadenylation control element that induces a a major pathway for mRNA degradation in S. cerevisiae.

3Ј end response from a 5Ј site. The alteration in poly(A) Both stable (e.g., PGK1 mRNA, t1/2 ≈ 45 min) and unstable tail length was suggested to reflect a dynamic equilib- (e.g., MFA2 mRNA, t1/2 ≈ 3.5 min) mRNAs decay along rium between adenylation and deadenylation in the cy- a pathway where 3Ј deadenylation is followed by 5Ј toplasm, which may favor elongated poly(A) tails for decapping and subsequent 5Ј-to-3Ј exonuclease degra- polysomal and partial deadenylation for nonpolysomal dation (Muhlrad and Parker, 1994; Muhlrad et al., 1994). mRNAs (Hentze). This exonuclease appears to represent the xrn1 gene The posttranscriptional IRE/IRP system constitutes product, because xrn1-deficient strains are enriched in the regulatory center for the maintenance of cellular iron deadenylated and decapped transcripts that, at least for homeostasis (Theil, 1994). The mRNAs encoding the two some mRNAs, eventually decay along a second 3Ј-to-5Ј mitochondrial citric acid cycle enzymes aconitase and pathway of degradation. A highly related pathway also succinate dehydrogenase (small subunit) have now leads to degradation of mRNAs bearing premature been found to be translationally regulated by IREs translational termination codons: without requirement (Kohler et al., 1995; Gray et al., 1996). Since IRP-1 is for prior deadenylation, the mRNAs are decapped and rapidly activated by reactive oxygen intermediates degraded in an xrn1-dependent manner (Muhlrad and (ROIs) originating from the mitochondrial respiratory Parker, 1994). In a genetic screen, R. Parker’s research chain (Pantopoulos et al., 1996), it was suggested that group (University of Arizona, Tucson), in collaboration the regulation of citric acid cycle enzymes could indicate with A. Stevens (Oak Ridge National Laboratories, Oak a novel regulatory role of the IRE/IRP system in balanc- Ridge, Tennessee), has identified mutations in genes ing ATP synthesis against the generation of hazardous required for decapping. The DCP1 gene appears to en- ROIs: IRP-1 might act as a response switch to excess code the decapping enzyme, or at least part of a decap- ROIs conferring negative regulation of the citric acid ping complex, since highly purified decapping activities cycle that fuels the respiratory chain (Hentze). from yeast cells capable of the release of m7GDP from .kDa protein 30ف Whereas the IRE-mediated effect on the poly(A) tail in capped transcripts contain this somatic cells is dispensable for altering the translational Dcp1p-deficient strains display a slow growth pheno- fate of the mRNA, J. Richter (Worcester Foundation, type, mRNA deadenylation at roughly wild-type rates, Shrewsbury, Massachusetts) illustrated the pivotal role and accumulation of capped transcripts for both stable played by regulated polyadenylation for translational and unstable mRNAs. In addition, PGK1 mRNA bearing control in germ line cells and during early development. a nonsense codon at position 103 is also stabilized in The binding of CPEB (Hake and Richter, 1994) to the Dcp1p-deficient strains, indicating that the same decap- cytoplasmic polyadenylation element (CPE) in the 3Ј ping enzyme is responsible for deadenylation-depen- UTRs of c-mos and other Xenopus mRNAs triggers the dent and deadenylation-independent decapping. This polyadenylation and subsequent translation of these suggests that differences in mRNA decay rates will be transcripts during oocyte maturation (Stebbins-Boaz et influenced by gene products that modulate the rates of al., 1996). A mouse CPEB homolog with 80% amino acid decapping by Dcp1p on different mRNAs. Two factors identity to the Xenopus protein has recently been cloned that modulate the rate of decapping of normal tran- (Richter) and is probably the regulatory factor that in- scripts may be the mrt1 and mrt3 gene products. Muta- duces murine c-mos mRNA polyadenylation, leading to tions in these genes do not affect the enzymatic activity the translation of c-mos and progression from meiosis of Dcp1p as assayed in crude extracts or decapping of I to meiosis II (Gebauer et al., 1994). The mechanism by mRNAs with premature translational stop codons, but which CPEB binding induces polyadenylation is still to modulate the rate of decapping of deadenylated tran- be defined, as is the pathway along which poly(A) tail scripts and significantly prolong their half lives. Parker elongation promotes 5Ј translational initiation. However, suggested that mrt1 and mrt3 might influence the mRNP in maturing Xenopusoocytes, CPE-directed polyadenyl- structure in a way that exposes deadenylated mRNAs ation is accompanied by 2Ј-O-ribose methylation of the to Dcp1p activity. two 5Ј terminal transcribed nucleotides. Mutations in the CPE or the hexanucleotide polyadenylation signal Conclusions and Perspectives that abolish polyadenylation also prevent cap ribose A lot of exciting new data were presented during the methylation and translational activation. Even when workshop and it is clear that considerable progress is polyadenylation occurs in oocytes treated with an ana- being made in unravelling the mechanisms involved in log inhibitor of methyltransferases, the resulting lack of RNA maturation, transport, and translation. Both genetic cap ribose methylation is paralleled by a lack of transla- and biochemical approaches continue to identify new tional activation (Kuge and Richter, 1995). Richter sug- genes and gene products involved in mRNA formation gested that cap ribose methylation may offer a competi- and function and are helping to characterize the ways tive advantage for polysomal recruitment during oocyte in which specific RNA sequences and structures are maturation and speculated that the biochemical link be- recognized. We can anticipate a very detailed descrip- tween 3Ј polyadenylation and 5Ј ribose methylation tion of the factors involved in RNA polyadenylation, Cell 970

splicing, export, translation, and stability to emerge over Bohmann, K., Ferreira, J., Santama, N., Weis, K., and Lamond, A.I. the next few years. This work on basic reaction mecha- (1995). Molecular analysis of the coiled body. J. Cell Science (Suppl.) nisms should also pave the way to a better understand- 19, 107–113. ing of posttranscriptional regulatory events involved in Bogerd, A.M., Hoffman, J.A., Amberg, D.C., Fink, G.R., and Davis, L.I. (1994). nup1 mutants exhibit pleiotropic defects in nuclear pore controlling gene expression during development and complex function. J. Cell Biol. 127, 319–332. differentiation. Several presentations during the work- Bogerd, H.P., Fridell, R.A., Madore, S., and Cullen, B.R. (1995). Iden- shop also illustrated the importance of RNA studies for tification of a novel cellular cofactor for the Rev/Rex class of retrovi- molecular medicine and in particular for understanding ral regulatory proteins. Cell 82, 485–494. strategies employed by viral pathogens. By continuing Burd, C., and Dreyfuss, G.(1994). Conserved structures and diversity to study how RNA viruses such as HIV, influenza, and of functions of RNA-binding proteins. Science 265, 615–621. picornaviruses subvert the splicing, export, and transla- Chang, D.D., and Sharp, P.A. (1989). Regulation by HIV Rev depends tion machineries, more important information will upon recognition of splice sites. Cell 59, 789–795. emerge about how these processes act on cellular RNAs Cullen, B.R. (1992). Mechanism of action of regulatory proteins en- in uninfected cells. coded by complex retroviruses. Microbiol. Rev. 56, 375–394. Another interesting theme that emerged during the Cullen, B.R., and Malim, M.H. (1991). The HIV-1 Rev protein: proto- workshop was the growing impact of the large scale type of a novel class of eukaryotic posttranscriptional regulators. sequencing projects that are currently underway and Trends Biochem. Sci. 16, 346–350. the utility of the rapidly expanding sequence databases. Daly, T.J., Cook, K.S., Gray, G.S., Maione, T.E., and Rusche, J.R. Here we can confidently predict that future studies in (1989). Specific binding of HIV-1 recombinant Rev protein to the the RNA field, as in most other areas of molecular biol- Rev-responsive element in vitro. Nature 342, 816–819. ogy, will benefit greatly from these resources. The identi- Davis, L.I. (1995). The nuclear pore complex. Annu. Rev. Biochem. fication and cloning of genes will be simplified, and 64, 865–896. structure/function comparisons of homologous proteins De Benedetti, A., and Rhoads, R.E. (1990). Overexpression of eukar- from different species should offer important insights yotic protein synthesis initiation factor 4E in HeLa cells results in aberrant growth and morphology. Proc. Natl. Acad. Sci. USA 87, into the mechanisms of RNA processing and transport. 8212–8216. However, despite the obvious advances that were Dreyfuss, G., Matunis, M.J., Pi˜ol-Roma, S., and Burd, C.G. (1993). reported during the workshop, in many cases the pre- hnRNP proteins and the biogenesis of mRNA. Annu. Rev. Biochem. sentations also underlined just how much remains to be 62, 289–321. learned about very basic aspects of gene expression Enami, K., Sato, T.A., Nakada, S., and Enami, M. (1994). Influenza and its regulation. For example, the data presented by virus NS1 protein stimulates translation of the M1 protein. J. Virol. C. Milstein, showing that in-frame stop codons are rec- 68, 1432–1437. ognized in the nucleus by a mechanism that is appar- Fischer, U., Huber, J., Boelens, W.C., Mattaj, I.W., and Lu¨ hrmann, ently independent of the translational machinery and R. (1995). The HIV-1 Rev activation domain is a nuclear export signal which causes an inhibition of splicing, demonstrate the that accesses an export pathway used by specific cellular RNAs. major gaps that remain in our understanding of how Cell 82, 475–483. RNA is recognized and how functional mRNAs are pro- Flach, J., Bossie, M., Vogel, J., Corbett, A., Jinks, T., Willins, D.A., duced.A similar conclusioncan be drawn from the unex- and Silver, P. (1994). A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm. Mol. Cell. Biol. 14, 8399–8407. pected discovery of diversity in the structure of spliceosomes reported by J. Steitz. The Juan March Workshop Fortes, P., Beloso, A., and Ortin,J. (1994). Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic illustrated the possibilities for work in the RNA field to transport. EMBO J. 13, 704–712. benefit from collaborations between disciplines such as Fritz, C.C., Zapp, M.L., and Green, M.R. (1995). A human nucleop- genetics, biochemistry, and virology. It is clear that this orin-like protein that specifically interacts with HIV Rev. Nature 376, now extends also into cell biology, as shown by the 530–533. talks on transport of macromolecules into and out of Fu, X.-D. (1995). The superfamily of arginine/serine-rich splicing fac- the nucleus and on nuclear organization. A major chal- tors. RNA 1, 663–680. lenge for future studies will involve defining the transi- Gebauer, F., Xu, W., Cooper, G.M., and Richter, J.D. (1994). Transla- tory steps that mRNAs make from processing to trans- tional control by cytoplasmic polyadenylation of c-mos mRNA is port to translation, as well as relating the detailed necessary for oocyte maturation in the mouse. EMBO J. 13, 5712– molecular data obtained from in vitro analyses with how 5720. these processes take place in the living cell. Gray, N.K., and Hentze, M.W. (1994). Iron regulatory protein prevents binding of the 43S translation pre-initiation complex to ferritin and eALAS mRNAs. EMBO J. 13, 3882–3891. References Gray, N.K., Pantopoulos, K., Dandekar, T., Ackrell, B.A.C., and Hen- Amberg, D.C., Goldstein, A.L., and Cole, C.N. (1992). Isolation and tze, M.W. (1996). Translational regulation of mammalian and dro- characterization of RAT1: an essential gene of Saccharomyces cere- sophila citric acid cycle enzymes via iron-responsive elements. visiae required for the efficient nucleocytoplasmic trafficking of Proc. Natl. Acad. Sci. USA 93, in press. mRNA. Genes Dev. 6, 1173–1189. Gorsch, L.C., Dockendorff, T.C., and Cole, C.N. (1995). A conditional Aoufouchi, S., Ye´lamos, J., and Milstein, C. (1996). Nonsense muta- allele of the novel repeat-containing yeast nucleoporin RAT7/ tions inhibit RNA splicing in a cell free system: recognition of mutant NUP159 causes both rapid cessation of mRNA export and reversible codon is independent of protein synthesis and tissue specific. Cell clustering of nuclear pore complexes. J. Cell Biol. 129, 939–955. 85, 415–422. Grandi, P., Emig, S., Weise, C., Hucho, F., Pohl, T., and Hurt, E.C. Beretta, L., Gingras, A.-C., Svitkin, Y.V., Hall, M.N., and Sonenberg, (1995). A novel nuclear pore protein Nup82p which specifically binds N. (1996). Rapamycin blocks the phosphorylation of 4E-BP1 and to a fraction of Nsp1p. J. Cell Biol. 130, 1263–1273. inhibits cap-dependent initiation. EMBO J. 15, 658–664. Guddat, U., Bakken, A.H., and Pieler, T. (1990). Protein-mediated Meeting Review 971

nuclear export of RNA: 5S rRNA containing small RNPs in Xenopus Martinez-Salas, E., Saiz, J.-C., Davila, M., Belsham, G.J., and Do- oocytes. Cell 60, 619–628. mingo, E. (1993). A single nucleotide substitution in the internal Haghighat, A., Mader, S., Pause, A., and Sonenberg, N. (1995). Re- ribosome entry site of foot-and-mouth disease virus leads to en- pressionof cap-dependent translation by 4E-binding protein 1: com- hanced cap-independent translation in vivo. J. Virol. 67, 3748–3755. petition with p220 for binding to eukaryotic initiation factor-4E. Melchior, F., Paschal, B., Evans, J., and Gerace, L. (1993). Inhibition EMBO J. 14, 5701–5709. of nuclear protein import by nonhydrolyzable analogues of GTP and Hake, L.E., and Richter, J.D. (1994). CPEB is a specificity factor identification of the small GTPase Ran/TC4 as an essential transport that mediates cytoplasmic polyadenylation during Xenopus oocyte factor. J. Cell Biol. 123, 1649–1659. maturation. Cell 79, 617–627. Mehlin, H., and Daneholt, B. (1993). The Balbiani ring particle: a model for the assembly and export of RNPs from the nucleus? Hall, S.L., and Padgett, R.A. (1994). Conserved sequences in a class Trends Cell Biol. 3, 443–447. of rare eukaryotic nuclear introns with non-consensus splice sites. J. Mol. Biol. 239, 357–365. Meyer, B.E., Meinkoth, J.L., and Malim, M.H. (1996). Nuclear transport of human immunodeficiency virus type 1, visna virus, and Hall, S.L., and Padgett, R.A. (1996). Requirement for U12 snRNA for equine infectious anemia virus Rev proteins: identification of a family in vivo splicing of a minor class of eukaryotic nuclear pre-mRNA of transferable nuclear export signals. J. Virol. 70, 2350–2359. introns. Science 271, 1716–1718. Michael, W.M., Choi, M., and Dreyfuss, G. (1995a). A nuclear export Izaurralde, E., and Mattaj, I.W. (1995). RNA export. Cell 81, 153–159. signal in hnRNP A1: a signal-mediated, temperature-dependent nu- Izaurralde, E., Lewis, J., McGuigan, C., Jankowska, M., Darzynkie- clear protein export pathway. Cell 83, 415–422. wicz, E., and Mattaj, I.W. (1994). A nuclear cap binding protein com- Michael, W.M., Siomi, H., Choi, M., Pin˜ ol-Roma, S., Nakielny, S., Liu, plex involved in pre-mRNA splicing. Cell 78, 657–668. Q., and Dreyfuss, G. (1995b). Signal sequences that target nuclear Izaurralde, E., Lewis, J., Gamberi, C., Jarmolowski, A., McGulgan, import and nuclear export of pre-mRNA-binding proteins. In Protein C., and Mattaj, I.W. (1995). A cap-binding protein complex mediating Kinesis. Cold Spring Harbor Symp. Quant. Biol. 60, 663–668. U snRNA export. Nature 376, 709–712. Moore, M.S., and Blobel, G. (1993). The GTP-binding protein Ran/ Kadowaki, T., Zhao, Y., and Tartakoff, A.M. (1992). A conditional TC4 is required for protein import into the nucleus. Nature 365, yeast mutant deficient in mRNA transport from nucleus to cyto- 661–663. plasm. Proc. Natl. Acad. Sci. USA 89, 2312–2316. Moore, J.M., Query, C.C., and Sharp, P.A. (1993). Splicing of precur- Kataoka, N., Ohno, M., Kangawa, K., Tokoro, Y., and Shimura, Y. sors to mRNAs by the spliceosome. In The RNA World, R.F. (1994). Cloning of a complementary DNA encoding an 80 kilodalton Gesteland and J.F. Atkins, eds. (Cold Spring Harbor, New York: Cold nuclear cap binding protein. Nucleic Acids Res. 22, 3861–3865. Spring Harbor Laboratory Press), pp. 303–358. Keller, W. (1995). No end yet to messenger RNA 3Ј processing! Cell Muhlrad, D., and Parker, R. (1994). Premature translational termina- 81, 829–832. tion triggers mRNA decapping. Nature 370, 578–581. Kohler, S.A., Henderson, B.R., and Kuhn, L.C. (1995). Succinate Muhlrad, D., Decker, C., and Parker, R. (1994). Deadenylation of the dehydrogenase b mRNA of Drosophila melanogaster has a func- unstable mRNA encoded by the yeast MFA2 gene leads to decap- tional iron-responsive element in its 5Ј-untranslated region. J. Biol. ping followed by 5Ј to 3Ј degradation of the transcript. Genes Dev. Chem. 270, 30781–30786. 8, 855–866. Kuge, H., and Richter, J.D. (1995). Cytoplasmic 3Ј poly(A) addition Nagai, K. (1996). RNA–protein interactions. Curr. Biol. 6, 53–61. induces 5Ј cap ribose methylation: implications for translational con- Nilsen, T.W. (1994). RNA–RNA interactions in the spliceosome: un- trol of maternal mRNA. EMBO J. 14, 6301–6310. ravelling the ties that bind. Cell 78, 1–4. Laird-Offringa, I.A., and Belasco, J.G. (1995). Analysis of RNA-bind- Ohno, M., Kataoka, N., and Shimura, Y. (1990). A nuclear cap binding ing proteins by in-vitro genetic selection of an amino acid residue protein from HeLa cells. Nucleic Acids Res. 18, 6989–6995. important for locking U1A onto RNA target. Proc. Natl. Acad. Sci. Pantopoulos, K., Weiss, G., and Hentze, M.W. (1996). Nitric oxide USA, 92, 11859–11863. and oxidative stress (H2O2) control mammalian iron metabolism by Lazaris-Karatzas, A., Montine, K.S., and Sonenberg, N. (1990). Ma- different pathways. Mol. Cell Biol., in press. lignant transformation by a eukaryotic initiation factor subunit that Pause, A., Belsham, G.J., Gingras, A.-C., Donze, O., Lin, T.-A., Law- binds to mRNA 5 cap. Nature 345, 544–547. Ј rence, J.C., and Sonenberg, N. (1994). Insulin-dependent stimulation Lefebvre, S., Burglen, L., Reboullet, S., Clermont, O., Burlet, P., of protein synthesis by phosphorylation of a regulator of 5Ј-cap Viollet, L., Benichou, B., Cruaud, C., Millasseau, P., Zeviani, M., et function. Nature 371, 762–767. al. (1995). Identification and characterization of a spinal muscular Pin˜ ol-Roma, S., and Dreyfuss, G. (1991). Transcription-dependent atrophy-determining gene. Cell 80, 155–165. and transcription-independent nuclear transport of hnRNP proteins. Legrain, P., and Rosbash, M. (1989). Some cis- and trans-acting Science 253, 312–314. mutants for splicing target pre-mRNA to the cytoplasm. Cell 57, Pin˜ ol-Roma, S., and Dreyfuss, G. (1992). Shuttling of pre-mRNA 573–583. binding proteins between nucleus and cytoplasm. Nature 355, Lin, T.-A., Kong, X., Haystead, T.A.J., Pause, A., Belsham, G., So- 730–732. nenberg, N., and Lawrence, J.C. (1994). PHAS-I as a link between Pin˜ ol-Roma, S., and Dreyfuss, G. (1993). hnRNP proteins: localiza- mitogen-activated protein kinase and translation initiation. Science tion and transport between the nucleus and the cytoplasm. Trends 266, 653–656. Cell Biol. 3, 151–155. Liu, Q., and Dreyfuss, G. (1996). A novel structure containing the Qiu, Y., Nemeroff, M., and Krug, R.M. (1995). The influenza virus survival of motor neurons protein. EMBO J., in press NS1 protein binds to specific region in human U6 snRNA and inhibits Lozano, F., Maertzdorf, B., Pannell, R., and Milstein, C. (1994). Low U6–U2 and U6–U4 snRNA interactions during splicing. RNA 1, cytoplasmic mRNA levels of immunoglobulin kappa light chain 304–316. genes containing nonsense codons correlate with inefficient splic- Russell, I.D., and Tollervey, D. (1992). NOP3 is an essential yeast ing. EMBO J. 13, 4617–4622. protein which is required for pre-rRNA processing. J. Cell Biol. 119, Lu, Y., Qian, X.Y., and Krug, R.M. (1994). The influenza NS1 protein: 737–747. a novel inhibitor of pre-mRNA splicing. Genes Dev. 8, 1817–1828. Sazer, S. (1996). The search for the primary function of the Ran Martin, G., and Keller, W. (1996). Mutational analysis of mammalian GTPase continues. Trends Cell Biol. 6, 81–85. poly(A) polymerase identifies a region for primer binding and a cata- Schneiter, R., Kadowaki, T., and Tartakoff, A.M. (1995). mRNA trans- lytic domain, homologous to the family X polymerases, and to other port in yeast: time to reinvestigate the functions of the nucleolus. nucleotidyltransferases. EMBO J., in press. Mol. Biol. Cell 6, 357–370. Cell 972

Schnierle, B.S., Gershon, P.D., and Moss, B. (1992). Cap-specific mRNA (nucleoside-O2Ј-)-methyltransferase and poly(A) polymerase stimulatory activities of vaccinia virus are mediated by a single protein. Proc. Natl. Acad. Sci. USA 89, 2897–2901. Sonenberg, N. (1996). mRNA 5Ј cap-binding protein eIF4E and control of cell growth. In Translational Control, Hershey, J.W.B., Ma- thews, M.B., and Sonenberg, N., eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), pp. 245–269. Stebbins-Boaz, B., Hake, L.E., and Richter, J.D. (1996). CPEB con- trols the cytoplasmic polyadenylation of cyclin, cdk2 and c-mos mRNAs and is necessary for oocyte maturation in Xenopus. EMBO J. 15, in press. Stripecke, R., Oliveira, C.C., McCarthy, J.E.G., and Hentze, M.W. (1994). Proteins binding to 5Ј untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells. Mol. Cell. Biol. 14, 5898–5909. Stutz, F., and Rosbash, M. (1994). A functional interaction between Rev and yeast pre-mRNA is related to splicing complex formation. EMBO J. 13, 4096–4104. Stutz, F., Neville, M., and Rosbash, M. (1995). Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast. Cell 82, 495–506. Sweet, D.J., and Gerace, L. (1995). Taking from the cytoplasm and givingto the pore: solubletransport factors in nuclear protein import. Trends Cell Biol. 5, 444–447. Tani, T., Derby, R.J., Hiraoka, Y., and Spector, D.L. (1995). Nucleolar accumulation of poly(A)ϩ RNA in heat-shocked yeast cells: implications of nucleolar involvement in messenger RNA transport. Mol. Biol. Cell 6, 1515–1534. Tarn, W.-Y., and Steitz, J. (1996). A novel spliceosome containing U11, U12, and U5 snRNPs excises a minor class (AT-AC) intron in vitro. Cell 84, 801–811. Theil, E.C. (1994). Iron regulatory elements (IREs): a family of mRNA non-coding sequences. Biochem. J. 304, 1–11. Visa, N., Alzhanova-Ericsson, A.T., Sun, X., Kiseleva, E., Bjo¨ rkroth, B. Wurtz, T., and Daneholt, B. (1996). A pre-mRNA-binding protein accompanies the RNA from the gene through the nuclear pores and into polysomes. Cell 84, 253–264. Wahle, E., and Keller, W. (1996). The biochemistry of polyadenylation. Trends Biochem. Sci., in press Wen, W., Meinkoth, J.L., Tsien, R.Y., and Taylor, S.S. (1995). Identifi- cation of a signal for rapid export of proteins from the nucleus. Cell 82, 463–473. Wente, S.R., and Blobel, G. (1994). NUP145 encodes a novel yeast glycine-leucine-phenylalanine-glycine (GLFG) nucleoporin required for nuclear envelope structure. J. Cell Biol. 125, 955–969. Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T., and Mann, M. (1996). Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466–469. Wilson, S.M., Datar, K.V., Paddy, M.R., Sedlow, J.R., and Swanson, M.S. (1994). Characterization of nuclear polyadenylated RNA-binding proteins in Saccharomyces cerevisiae. J. Cell Biol. 127, 1173– 1184. Zapp, M.L., and Green, M.R. (1989). Sequence-specific RNA binding by the HIV-1 Rev protein. Nature 342, 714–716.