Mapping of sequences in the 5' region and 3' UTR of tomato ringspot virus RNA2 that facilitate cap-independent translation of reporter transcripts in vitro.

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5'-viral genome-linked protein (VPg) and have a 3' polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3' untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5' regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5' region and 3' UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5' region and 3' UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5' proximal coding region of the RNA2 polyprotein and in the RNA2 3' UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3' UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3' UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5' region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773-972) of the 1547 nt long 3' UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.

Retention of exogenous mRNAs selectively in the germ cells of the sea urchin requires only a 5'-cap and a 3'-UTR.

The abundance of an mRNA in a cell depends on its overall rates of synthesis and decay. RNA stability is an important element in the regulation of gene expression, and is achieved by a variety of processes including specific recruitment of nucleases and RNAi-associated mechanisms. These mechanisms are particularly important in stem cells, which, in many cases, have attenuated transcription. Here we report that exogenous mRNA injected into fertilized eggs of the sea urchin is selectively retained in the small micromeres, which contribute to the germ line in this organism, beginning in blastulae, when compared to adjacent somatic cells. We show that modification of this exogenous RNA using cap analogs and poly-adenosine tail deletions do not affect its selective retention in the small micromeres, but removal of the cap or of the 3'-untranslated region eliminates any selective mRNA retention in the presumptive germ line. Our results illuminate a likely ancient mechanism used by stem cells to prolong the lifespan of RNAs-either through RNA protection or by the absence of basic RNA degradation mechanisms, which are employed by most other cells of an organism.

Activated 4E-BP1 represses tumourigenesis and IGF-I-mediated activation of the eIF4F complex in mesothelioma.

BACKGROUND: Insulin-like growth factor (IGF)-I signalling stimulates proliferation, survival, and invasion in malignant mesothelioma and other tumour types. Studies have found that tumourigenesis is linked to dysregulation of cap-dependent protein translation. METHODS: The effect of IGF stimulation on cap-mediated translation activation in mesothelioma cell lines was studied using binding assays to a synthetic 7-methyl GTP-cap analogue. In addition, cap-mediated translation was genetically repressed in these cells with a dominant active motive of 4E-BP1. RESULTS: In most mesothelioma cell lines, IGF-I stimulation resulted in a hyperphosphorylation-mediated inactivation of 4E-BP1 compared with that in normal mesothelial cells. An inhibitor of Akt diminished IGF-I-mediated phosphorylation of 4E-BP1, whereas inhibiting MAPK signalling had no such effect. IGF-I stimulation resulted in the activation of the cap-mediated translation complex as indicated by an increased eIF4G/eIF4E ratio in cap-affinity assays. Akt inhibition reversed the eIF4G/eIF4E ratio. Mesothelioma cells transfected with an activated 4E-BP1 protein (4E-BP1(A37/A46)) were resistant to IGF-I-mediated growth, motility, and colony formation. In a murine xenograft model, mesothelioma cells expressing the dominant active 4E-BP1(A37/A46) repressor protein showed abrogated tumourigenicity compared with control tumours. CONCLUSION: IGF-I signalling in mesothelioma cells drives cell proliferation, motility, and tumourigenesis through its ability to activate cap-mediated protein translation complex through PI3K/Akt/mTOR signalling.

YB-1 translational control of epithelial-mesenchyme transition.

Transitions between epithelial and mesenchmal phenotypes play critical roles in normal development and cancer progression. In this issue of Cancer Cell, Evdokimova et al. demonstrate that YB-1 regulates epithelial-mesenchyme transition (EMT) by inducing cap-independent translation of mRNAs encoding EMT-promoting factors and suppressing cap-dependent translation of mRNAs encoding growth-promoting factors.

Use of in vitro translation extract depleted in specific initiation factors for the investigation of translational regulation.

Regulation of gene expression often involves the control of translation mediated through one or more initiation factors that are required for the translation of eukaryotic mRNAs. Genetic and molecular biological approaches can be highly useful in the initial identification of translational regulation, but the use of in vitro translation lysates can be essential in elucidating the details of translational regulatory mechanisms. Wheat germ lysate has long been used for in vitro translation studies. The noncompetitive conditions that prevail in this lysate as it is normally produced, however, preclude the translational regulatory analysis of many mRNAs involving the preferential recruitment of initiation factors. The development of lysate depleted in specific translation initiation factors converts wheat germ lysate from a noncompetitive system to one that is competitive in a fast and simple procedure that enables it to be used in the analysis of many more translational regulatory mechanisms than is currently possible with unfractionated lysate.

MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F.

MicroRNAs (miRNAs) play an important role in gene regulatory networks in animals. Yet, the mechanistic details of their function in translation inhibition or messenger RNA (mRNA) destabilization remain controversial. To directly examine the earliest events in this process, we have developed an in vitro translation system using mouse Krebs-2 ascites cell-free extract that exhibits an authentic miRNA response. We show here that translation initiation, specifically the 5' cap recognition process, is repressed by endogenous let-7 miRNAs within the first 15 minutes of mRNA exposure to the extract when no destabilization of the transcript is observed. Our results indicate that inhibition of translation initiation is the earliest molecular event effected by miRNAs. Other mechanisms, such as mRNA degradation, may subsequently consolidate mRNA silencing.

Quantitative proteomics identifies Gemin5, a scaffolding protein involved in ribonucleoprotein assembly, as a novel partner for eukaryotic initiation factor 4E.

Protein complexes are dynamic entities; identification and quantitation of their components is critical in elucidating functional roles under specific cellular conditions. We report the first quantitative proteomic analysis of the human cap-binding protein complex. Components and proteins associated with the translation initiation eIF4F complex that may affect complex formation were identified and quantitated under distinct growth conditions. Site-specific phosphorylation of eIF4E and eIF4G and elevated levels of eIF4G:eIF4E complexes in phorbol ester treated HEK293 cells, and in serum-starved tumorigenic human mesenchymal stromal cells, attested to their activated translational states. The WD-repeat, scaffolding-protein Gemin5 was identified as a novel eIF4E binding partner, which interacted directly with eIF4E through a motif (YXXXXLPhi) present in a number of eIF4E-interacting partners. Elevated levels of Gemin5:eIF4E complexes were found in phorbol ester treated HEK293 cells. Gemin5 and eIF4E co-localized to cytoplasmic P-bodies in human osteosarcoma U2OS cells. Interaction between eIF4E and Gemin5 and their co-localization to the P-bodies, may serve to recruit capped mRNAs to these RNP complexes, for functions related to RNP assembly, remodeling and/or transition from active translation to mRNA degradation. Our results demonstrate that our quantitative proteomic strategy can be applied to the identification and quantitation of protein complex components in human cells grown under different conditions.

Cap-independent translation mechanism of red clover necrotic mosaic virus RNA2 differs from that of RNA1 and is linked to RNA replication.

The genome of Red clover necrotic mosaic virus (RCNMV) in the genus Dianthovirus is divided into two RNA molecules of RNA1 and RNA2, which have no cap structure at the 5' end and no poly(A) tail at the 3' end. The 3' untranslated region (3' UTR) of RCNMV RNA1 contains an essential RNA element (3'TE-DR1), which is required for cap-independent translation. In this study, we investigated a cap-independent translational mechanism of RNA2 using a firefly luciferase (Luc) gene expression assay system in cowpea protoplasts and a cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts. We were unable to detect cis-acting RNA sequences in RNA2 that can replace the function of a cap structure, such as the 3'TE-DR1 of RNA1. However, the uncapped reporter RNA2, RNA2-Luc, in which the Luc open reading frame (ORF) was inserted between the 5' UTR and the movement protein ORF, was effectively translated in the presence of p27 and p88 in protoplasts in which RNA2-Luc was replicated. Time course experiments in protoplasts showed that the translational activity of RNA2-Luc did not reflect the amount of RNA2. Mutations in cis-acting RNA replication elements of RNA2 abolished the cap-independent translational activity of RNA2-Luc, suggesting that the translational activity of RNA2-Luc is coupled to RNA replication. Our results show that the translational mechanism differs between two segmented genomic RNAs of RCNMV. We present a model in which only RNA2 that is generated de novo through the viral RNA replication machinery functions as mRNA for translation.

Calcium signaling stimulates translation of HIF-alpha during hypoxia.

Hypoxia-inducible factors (HIFs) are ubiquitous transcription factors that mediate adaptation to hypoxia by inducing specific sets of target genes. It is well accepted that hypoxia induces accumulation and activity of HIFs by causing stabilization of their alpha subunits. We have demonstrated that hypoxia stimulates translation of HIF-1alpha and -2alpha proteins by distributing HIF-alpha mRNAs to larger polysome fractions. This requires influx of extracellular calcium, stimulation of classical protein kinase C-alpha (cPKC-alpha), and the activity of mammalian target of rapamycin, mTOR. The translational component contributes to approximately 40-50% of HIF-alpha proteins accumulation after 3 h of 1% O2. Hypoxia also inhibits general protein synthesis and mTOR activity; however, cPKC-alpha inhibitors or rapamycin reduce mTOR activity and total protein synthesis beyond the effects of hypoxia alone. These data show that during general inhibition of protein synthesis by hypoxia, cap-mediated translation of selected mRNAs is induced through the mTOR pathway. We propose that calcium-induced activation of cPKC-alpha hypoxia partially protects an activity of mTOR from hypoxic inhibition. These results provide an important physiologic insight into the mechanism by which hypoxia-stimulated influx of calcium selectively induces the translation of mRNAs necessary for adaptation to hypoxia under conditions repressing general protein synthesis.

Unrip, a factor implicated in cap-independent translation, associates with the cytosolic SMN complex and influences its intracellular localization.

Spliceosomal Uridine-rich small ribonucleo protein (U snRNP) assembly is an active process mediated by the macromolecular survival motor neuron (SMN) complex. This complex contains the SMN protein and six additional proteins, named Gemin2-7, according to their localization to nuclear structures termed gems. Here, we provide biochemical evidence for the existence of another, yet atypical, SMN complex component, termed unr-interacting protein (unrip). This abundant factor has been previously shown to form a complex with unr, a protein implicated in cap-independent translation of cellular and viral mRNA. We show that unrip is integrated into a complex with unr or with the SMN complex in vivo in a mutually exclusive manner. In the latter case, unrip is recruited to the active SMN complex via a stable interaction with Gemin7. However, unlike SMN and Gemins, unrip localizes predominantly to the cytoplasm and is absent from gems/Cajal bodies. Interestingly, RNAi-induced reduction of unrip protein levels leads to enhanced accumulation of SMN in the nucleus as evident by the increased formation of nuclear gems/Cajal bodies. Our data identify unrip as the first component of the U snRNP assembly machinery that associates with the SMN complex in a compartment-specific way. We speculate that unrip plays a crucial role in the intracellular distribution of the SMN complex.

An internal ribosome entry site mediates translation of lymphoid enhancer factor-1.

The lymphoid enhancer factor-1 LEF1 locus produces multiple mRNAs via alternative promoters. Full-length LEF-1 protein is produced via translation of an mRNA with a 1.2-kb, GC-rich 5'-untranslated region (UTR), whereas a truncated LEF-1 isoform is produced by an mRNA with a short, 60-nucleotide (nt) 5'-UTR. Full-length LEF-1 promotes cell growth via its interaction with the WNT signaling mediator beta-catenin. Truncated LEF-1 lacks the beta-catenin binding domain and opposes WNT signaling as a competitive inhibitor for WNT response elements. In this study we tested the hypothesis that the long, GC-rich 5'-UTR within the full-length LEF1 mRNA contains an internal ribosome entry site (IRES). Using a dicistronic vector in transient DNA transfections, we show that the LEF1 5'-UTR mediates cap-independent translation. Additional experiments involving a promoter-less dicistronic vector, Northern blot analysis, and transient transfections of dicistronic mRNAs into cultured mammalian cells compromised for cap-dependent translation demonstrate that the 5'-UTR of full-length LEF1 mRNA contains a bona fide IRES. Deletion analysis of the 5'-UTR shows that maximal IRES activity requires the majority of the 5'-UTR, consistent with the notion that cellular IRESs require multiple modules for efficient activity. This study demonstrates that full-length LEF1 mRNA has evolved to utilize a cap-independent mechanism for translation of full-length LEF-1, whereas the truncated isoform is produced via the canonical cap-dependent ribosome scanning mechanism.

A shine-dalgarno-like sequence mediates in vitro ribosomal internal entry and subsequent scanning for translation initiation of coxsackievirus B3 RNA.

Translation initiation of coxsackievirus B3 (CVB3) RNA is directed by an internal ribosome entry site (IRES) within the 5' untranslated region. However, the details of ribosome-template recognition and subsequent translation initiation are still poorly understood. In this study, we have provided evidence to support the hypothesis that 40S ribosomal subunits bind to CVB3 RNA via basepairing with 18S rRNA in a manner analogous to that of the Shine-Dalgarno (S-D) sequence in prokaryotic systems. We also identified a new site within both the 18S rRNA and the polpyrimidine-tract sequence of the IRES that allows them to form stronger sequence complementation. All these data were obtained from in vitro translation experiments using mutant RNAs containing either an antisense IRES core sequence at the original position or site-directed mutations or deletions in the polypyrimidine tract of the IRES. The mutations significantly reduced translation efficiency but did not abolish protein synthesis, suggesting that the S-D-like sequence is essential, but not sufficient for ribosome binding. To determine how ribosomes reach the initiation codon after internal entry, we created additional mutants: when the authentic initiation codon at nucleotide (nt) 742 was mutated, a 180-nt downstream in-frame AUG codon at nt 922 is able to produce a truncated smaller protein. When this mutation was introduced into the full-length cDNA of CVB3, the derived viruses were still infectious. However, their infectivity was much weaker than that of the wild-type CVB3. In addition, when a stable stem-loop was inserted upstream of the initiation codon in the bicistronic RNA, translation was strongly inhibited. These data suggest that ribosomes reach the initiation codon from the IRES likely by scanning along the viral RNA.

Cap-independent translation conferred by the 5' leader of tobacco etch virus is eukaryotic initiation factor 4G dependent.

The 5' leader of tobacco etch virus (TEV) genomic RNA directs efficient translation from the naturally uncapped viral mRNA. Two distinct regions within the TEV 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mRNA, indicating that the TEV leader contains an internal ribosome entry site (IRES). In this study, the requirements for TEV IRES activity were investigated. The TEV IRES enhanced translation of monocistronic or dicistronic mRNAs in vitro under competitive conditions, i.e., at high RNA concentration or in lysate partially depleted of eukaryotic initiation factor 4F (eIF4F) and eIFiso4F, the two cap binding complexes in plants. The translational advantage conferred by the TEV IRES under these conditions was lost when the lysate reduced in eIF4F and eIFiso4F was supplemented with eIF4F (or, to a lesser extent, eIFiso4F) but not when supplemented with eIF4E, eIFiso4E, eIF4A, or eIF4B. eIF4G, the large subunit of eIF4F, was responsible for the competitive advantage conferred by the TEV IRES. TEV IRES activity was enhanced moderately by the poly(A)-binding protein. These observations suggest that the TEV IRES directs cap-independent translation through a mechanism that involves eIF4G specifically.

The p36 isoform of BAG-1 is translated by internal ribosome entry following heat shock.

BAG-1 (also known as RAP46/HAP46) was originally identified as a 46 kDa protein that bound to and enhanced the anti-apoptotic properties of Bcl-2. BAG-1 exists as three major isoforms (designated p50, p46 and p36 or BAG-1L, BAG-1M and BAG-1S respectively) and one minor isoform (p29), which are translated from a common transcript. The differing amino terminus determines both the intracellular location and the repertoire of binding partners of the isoforms which play different roles in a variety of cellular processes including signal transduction, heat shock, apoptosis and transcription. Although in vitro data suggest that the four BAG-1 isoforms are translated by leaky scanning, the patterns of isoform expression in vivo, especially in transformed cells, do not support this hypothesis. We have performed in vivo analysis of the BAG-1 5' untranslated region and shown that translation initiation of the most highly expressed isoform (p36/BAG-1S) can occur by both internal ribosome entry and cap-dependent scanning. Following heat shock, when there is a downregulation of cap-dependent translation, the expression of the p36 isoform of BAG-1 is maintained by internal ribosome entry.

Core protein-coding sequence, but not core protein, modulates the efficiency of cap-independent translation directed by the internal ribosome entry site of hepatitis C virus.

Among a myriad of putative functions assigned to the hepatitis C virus (HCV) core protein, several studies suggest that it may modulate internal ribosome entry site (IRES)-mediated initiation of translation. We compared the translational activity of dicistronic reporter transcripts containing the HCV IRES within the intercistronic space fused to downstream sequence encoding either 22 amino acids (aa) or 173 aa of the core protein. The inclusion of the nearly full-length core protein-coding sequence significantly suppressed translation in vitro and in transfected HepG2 cells. However, this suppression was not eliminated by frameshift mutations introduced into the core sequence, suggesting that it occurred at the RNA level and not as a result of core protein expression in cis. Similarly, the expression of core protein (aa 1 to 191) in trans from a recombinant baculovirus did not suppress IRES-directed translation from any of these transcripts in transfected Huh-7 cells. While core protein expression did decrease IRES activity in HepG2 cells (up to 79% suppression), the expression of beta-galactosidase from a control baculovirus also suppressed IRES activity (up to 56%), strongly suggesting that this suppression was nonspecific. Finally, the addition of purified recombinant core protein (aa 1 to 179) to in vitro translation reactions at concentrations up to a 10-fold molar excess over the RNA transcripts resulted in no significant reduction in IRES activity. Consistent with these results, a gel retention assay indicated no difference in the affinities of the recombinant HCV core protein and a recombinant Venezuelan equine encephalitis virus capsid protein for HCV IRES-containing RNA transcripts. We conclude that while the inclusion of core protein-coding sequence downstream of the IRES may reduce the efficiency of cap-independent translation on HCV RNA, the core protein itself has no biologically relevant activity in modulating HCV IRES activity.

Internal ribosome initiation of translation and the control of cell death.

The majority of cellular stresses lead to the inhibition of cap-dependent translation. Some mRNAs, however, are translated by a cap-independent mechanism, mediated by ribosome binding to internal ribosome entry site (IRES) elements located in the 5' untranslated region. Interestingly, IRES elements are found in the mRNAs of several survival factors, oncogenes and proteins crucially involved in the control of apoptosis. These mRNAs are translated under a variety of stress conditions, including hypoxia, serum deprivation, irradiation and apoptosis. Thus, IRES-mediated translational control might have evolved to regulate cellular responses in acute but transient stress conditions that would otherwise lead to cell death.

Downstream ribosomal entry for translation of coronavirus TGEV gene 3b.

Gene 3b (ORF 3b) in porcine transmissible gastroenteritis coronavirus (TGEV) encodes a putative nonstructural polypeptide of 27.7 kDa with unknown function that during translation in vitro is capable of becoming a glycosylated integral membrane protein of 31 kDa. In the virulent Miller strain of TGEV, ORF 3b is 5'-terminal on mRNA 3-1 and is presumably translated following 5' cap-dependent ribosomal entry. For three other strains of TGEV, the virulent British FS772/70 and Taiwanese TFI and avirulent Purdue-116, mRNA species 3-1 is not made and ORF 3b is present as a non-overlapping second ORF on mRNA 3. ORF 3b begins at base 432 on mRNA 3 in Purdue strain. In vitro expression of ORF 3b from Purdue mRNA 3-like transcripts did not fully conform to a predicted leaky scanning pattern, suggesting ribosomes might also be entering internally. With mRNA 3-like transcripts modified to carry large ORFs upstream of ORF 3a, it was demonstrated that ribosomes can reach ORF 3b by entering at a distant downstream site in a manner resembling ribosomal shunting. Deletion analysis failed to identify a postulated internal ribosomal entry structure (IRES) within ORF 3a. The results indicate that an internal entry mechanism, possibly in conjunction with leaky scanning, is used for the expression of ORF 3b from TGEV mRNA 3. One possible consequence of this feature is that ORF 3b might also be expressed from mRNAs 1 and 2.

Regulation of the rapamycin and FKBP-target 1/mammalian target of rapamycin and cap-dependent initiation of translation by the c-Abl protein-tyrosine kinase.

The c-Abl protein-tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. The rapamycin and FKBP-target 1 (RAFT1), also known as FKBP12-rapamycin-associated protein (FRAP, mTOR), regulates the p70S6 kinase (p70(S6k)) and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). The present results demonstrate that c-Abl binds directly to RAFT1 and phosphorylates RAFT1 in vitro and in vivo. c-Abl inhibits autophosphorylation of RAFT1 and RAFT1-mediated phosphorylation p70(S6k). The functional significance of the c-Abl-RAFT1 interaction is further supported by the finding that eIF4E-dependent translation in mouse embryo fibroblasts from Abl(-/-) mice is significantly higher than that compared in wild-type cells. The results also demonstrate that exposure of cells to ionizing radiation is associated with c-Abl-mediated binding of 4E-BP1 to eIF4E and inhibition of translation. These findings with the c-Abl tyrosine kinase represent the first demonstration of a negative physiologic regulator of RAFT1-mediated 5' cap-dependent translation.

A novel form of DAP5 protein accumulates in apoptotic cells as a result of caspase cleavage and internal ribosome entry site-mediated translation.

Death-associated protein 5 (DAP5) (also named p97 and NAT1) is a member of the translation initiation factor 4G (eIF4G) family that lacks the eIF4E binding site. It was previously implicated in apoptosis, based on the finding that a dominant negative fragment of the protein protected against cell death. Here we address its function and two distinct levels of regulation during apoptosis that affect the protein both at translational and posttranslational levels. DAP5 protein was found to be cleaved at a single caspase cleavage site at position 790, in response to activated Fas or p53, yielding a C-terminal truncated protein of 86 kDa that is capable of generating complexes with eIF4A and eIF3. Interestingly, while the overall translation rate in apoptotic cells was reduced by 60 to 70%, in accordance with the simultaneous degradation of the two major mediators of cap-dependent translation, eIF4GI and eIF4GII, the translation rate of DAP5 protein was selectively maintained. An internal ribosome entry site (IRES) element capable of directing the translation of a reporter gene when subcloned into a bicistronic vector was identified in the 5' untranslated region of DAP5 mRNA. While cap-dependent translation from this transfected vector was reduced during Fas-induced apoptosis, the translation via the DAP5 IRES was selectively maintained. Addition of recombinant DAP5/p97 or DAP5/p86 to cell-free systems enhanced preferentially the translation through the DAP5 IRES, whereas neutralization of the endogenous DAP5 in reticulocyte lysates by adding a dominant negative DAP5 fragment interfered with this translation. The DAP5/p86 apoptotic form was more potent than DAP5/p97 in these functional assays. Altogether, the data suggest that DAP5 is a caspase-activated translation factor which mediates cap-independent translation at least from its own IRES, thus generating a positive feedback loop responsible for the continuous translation of DAP5 during apoptosis.

The nuclear cap-binding complex is a novel target of growth factor receptor-coupled signal transduction.

In an attempt to further understand how nuclear events (such as gene expression, nuclear import/export, and cell cycle checkpoint control) might be subject to regulation by extracellular stimuli, we sought to identify nuclear activities under growth factor control. Using a sensitive photoaffinity labeling assay that measured [alpha-32P]GTP incorporation into nuclear proteins, we identified the 20-kDa subunit of the nuclear cap-binding complex (CBC) as a protein whose binding activity is greatly enhanced by the extracellular stimulation of serum-arrested cells. The CBC represents a 20- and 80-kDa heterodimer (the subunits independently referred to as CBP20 and CBP80, respectively) that binds the 7-methylguanosine cap on RNAs transcribed by RNA polymerase II. This binding facilitates precursor messenger RNA splicing and export. We have demonstrated that the [alpha-32P]GTP incorporation into CBP20 was correlated with an increased ability of the CBC to bind capped RNA and have used the [alpha-32P]GTP photoaffinity assay to characterize the activation of the CBC in response to growth factors. We show that the CBC is activated by heregulin in HeLa cells and by nerve growth factor in PC12 cells as well as during the G1/S phase of the cell cycle and when cells are stressed with UV irradiation. Additionally, we show that cap-dependent splicing of precursor mRNA, a functional outcome of CBC activation, can be catalyzed by growth factor addition to serum-arrested cells. Taken together, these data identify the CBC as a nuclear target for growth factor-coupled signal transduction and suggest novel mechanisms by which growth factors can influence gene expression and cell growth.