HeLa Cell Factor Research Articles

Adrenocorticotropin/3',5'-cyclic AMP-mediated transcription of the scavenger akr1-b7 gene in adrenocortical cells is dependent on three functionally distinct steroidogenic factor-1-responsive elements.

The akr1-b7 gene encodes a scavenger enzyme expressed in steroidogenic glands under pituitary control. In the zona fasciculata of the adrenal cortex where its expression is controlled by ACTH, AKR1-B7 detoxifies isocaproaldehyde produced during the first step of steroidogenesis. Three steroidogenic factor-1 (SF-1)-responsive elements (SFREs) are contained within the -510/+41 promoter region, which was previously demonstrated to drive gene expression in transgenic mice adrenal cortex. All these sequences bind at least SF-1 in Y1 adrenocortical cell nuclear extracts and can be activated by overexpression of this factor in HeLa cells. However, the three SFREs show distinct properties regarding akr1-b7 promoter activity in Y1 cells. Whereas the proximal -102 SFRE supports basal promoter activity, the -458 bona fide SFRE is essential for both basal promoter activity and cAMP responsiveness, although it is unresponsive to cAMP when isolated from its promoter context. This suggests that SF-1 is not a cAMP-responsive factor per se. The neighboring SFRE at -503 is a palindromic sequence that binds monomeric and heteromeric SF-1 as well as an adrenal-specific complex. Using MA-10 Leydig cells and Y1-10r9 mutant cells, we provide evidence that its activity in adrenocortical cells depends on the binding of the adrenal-specific factor, which is required for basal and cAMP-induced promoter activity. Furthermore, the -503 site has intrinsic cAMP-sensing ability in Y1 cells, which is correlated with increased adrenal-specific complex binding. Collectively, our results suggest that cAMP responsiveness of the akr1-b7 promoter is achieved through cooperation between the adrenal-specific factor bound to the -503 site and SF-1 bound to the -458 site.

Complexes between the nonsense-mediated mRNA decay pathway factor human upf1 (up-frameshift protein 1) and essential nonsense-mediated mRNA decay factors in HeLa cells.

mRNAs harbouring premature translation-termination codons are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway. Human up-frameshift protein 1 (Hupf1) is an NMD factor that is conserved between yeast and mammals. To isolate cellular complexes that are formed with Hupf1 and to explore the role of cellular proteins in NMD, we generated a HeLa cell line that stably expresses Hupf1 bearing a double-affinity tag (termed Hupf1-2tag). Hupf1-2tag is localized in the cytoplasm similar to the endogenous Hupf1 protein, and the Hupf1-2tag cell line is fully NMD-competent. Using affinity chromatography, Hupf1-2tag-associated proteins were isolated. MS and immunoblotting identified the NMD factors Hupf2 and Hupf3a/b as interaction partners of Hupf1. Size-exclusion chromatography indicates that the NMD factors Hupf1, Hupf2 and the large isoform of Hupf3a might exist in a stable, high-molecular-mass complex of approx. 1.3 MDa. Interestingly, the poly(A)-binding protein was also identified by MS to be associated specifically with Hupf1-2tag. In contrast with the interaction with Hupf2 and Hupf3a/b, the association of poly(A)-binding protein with Hupf1 is highly sensitive to treatment of the isolated complexes with RNase. Components of the exon-exon junction complex or the translational eukaryotic release factor (eRF) 3 were not identified in complexes associated with Hupf1-2tag. We discuss these findings in the context of current models of NMD.

Selective interaction between the chromatin-remodeling factor BRG1 and the heterochromatin-associated protein HP1alpha.

Mammalian heterochromatin protein 1 (HP1) alpha, HP1beta and HP1gamma are closely related non-histone chromosomal proteins that function in gene silencing, presumably by organizing higher order chromatin structures. Here, we show by co-immunoprecipitation that HP1alpha, but neither HP1beta nor HP1gamma, forms a complex with the BRG1 chromatin-remodeling factor in HeLa cells. In vitro, BRG1 interacts directly and preferentially with HP1alpha. The region conferring this preferential binding has been mapped to residues 106-180 of the HP1alpha C-terminal chromoshadow domain. Using site-directed mutagenesis, we have identified three amino acid residues I113, A114 and C133 in HP1alpha (K, P and S in HP1beta and HP1gamma) that are essential for the selective interaction of HP1alpha with BRG1. Interestingly, these residues were also shown to be critical for the silencing activity of HP1alpha. Taken together, these results demonstrate that mammalian HP1 proteins are biochemically distinct and suggest an entirely novel function for BRG1 in modulating HP1alpha-containing heterochromatic structures.

Transcriptional regulation of the KEL gene and Kell protein expression in erythroid and non-erythroid cells

The Kell blood-group antigen was originally reported to be a protein expressed in erythroid tissue only. Transcriptional analysis of the KEL promoter activity in human erythroleukaemia K562 and epithelial HeLa cells by electrophoretic mobility-shift and supershift assays, chloramphenicol acetyltransferase assays, co-transfection studies and site-directed mutagenesis provided the following results: (i) the KEL promoter exhibits a strong transcriptional activity in K562 cells and, unexpectedly, a basal non-erythroid activity in HeLa cells, (ii) up-regulation of the 5′ distal promoter activity occurs only in the erythroid context, and (iii) two motifs localized in the exon 1 region, which bind the Sp1/Sp3 and the human GATA-1/Ku70/80 factors, were required for down-regulation of the promoter activity, but inhibition of the promoter activity by the repressing factors in HeLa cells was incomplete. KEL expression in HeLa cells was performed further by primer-extension analysis, which revealed the presence of a low amount of Kell transcript correlating with basal expression of the Kell protein in these cells, as shown by immunopurification and Western-blot analysis. DNA sequencing of the transcript revealed a sequence identical to that obtained from erythroid tissue. In human tissues, KEL expression was investigated by dot-blot analysis and revealed high levels of Kell mRNAs, particularly in brain tissues, testis and lymphoid tissues. Moreover, most tissues analysed exhibited low levels of Kell transcripts. The Kell protein was also detected by immunohistochemistry in the Sertoli cells of the testis and in lymphoid tissues like spleen and tonsil, specifically localized in the follicular dendritic cells. Altogether, the results indicated that KEL expression is not restricted to erythroid tissue.

Human T-lymphotropic Virus Type I Tax Protein Utilizes Distinct Pathways for p53 Inhibition That Are Cell Type-dependent

p53 plays a pivotal role in transmitting signals from many forms of genotoxic stress to genes and factors that control the cell cycle and apoptosis. We have previously shown that the human T-lymphotropic virus type I Tax protein can inhibit p53 function. Recently we reported that Tax inhibits p53 function in Jurkat cells and mouse embryo fibroblasts through a mechanism involving the nuclear factor kappa B pathway and correlates with phosphorylation on serines 15 and 392 of p53. However, several groups have also observed a mechanism that correlates with p300 binding of Tax. To address this controversy and to determine the mechanism by which Tax inhibits p53 function, we examined the activation functions of Tax required for p53 inhibition. In HeLa and H1299 cells the cAMP-response element-binding protein/activating transcription factor activation function is essential, as demonstrated by the Tax mutants M47 and K88A. In addition, expression of exogenous p300 in H1299 cells allows full recovery of p53 transactivation in the presence of Tax. Consistent with p300 being a limiting factor in H1299, Saos-2, and HeLa cells, we found that the level of endogenous p300 is relatively low in these cells compared with Jurkat cells or the human T-lymphotropic virus type I-infected C81 and MT2 cells. Thus our data suggests that Tax utilizes distinct mechanisms to inhibit p53 function that are cell type-dependent.

Polypyrimidine-tract binding protein (PTB) is necessary, but not sufficient, for efficient internal initiation of translation of human rhinovirus-2 RNA.

Initiation of translation of the animal picornavirus RNAs is via a mechanism of direct internal ribosome entry, which requires a substantial segment of the viral 5'-untranslated region, generally known as the IRES (for "internal ribosome entry site"). Because, however, translation of the RNAs of members of the enterovirus, and more especially, the rhinovirus subgroups of the Picornaviridae is restricted in the reticulocyte lysate system, but is greatly stimulated by the addition of HeLa cell extracts, the implication is that, in these cases, internal initiation also requires cellular trans-acting factors that are more abundant in HeLa cell extracts than in rabbit reticulocytes. This was used as the basis of a functional assay for the purification of the HeLa cell factors required for translation dependent on the human rhinovirus-2 (HRV) IRES. There are two such HeLa cell factors separable by ion-exchange chromatography, each of which is individually active in the assay, although their combined effect is synergistic. One of these activities is shown to be polypyrimidine-tract binding protein (PTB) on the grounds that (1) the activity copurifies to homogeneity with PTB and (2) recombinant PTB expressed in Escherichia coli stimulates HRV IRES-dependent translation with a specific activity similar to that of the purified HeLa cell factor. Furthermore, it is shown that recombinant PTB also stimulates the translation of RNAs bearing the poliovirus type 1 (Mahoney) IRES.

High-mobility group 1/2 proteins are essential for initiating rolling-circle-type DNA replication at a parvovirus hairpin origin.

Rolling-circle replication is initiated by a replicon-encoded endonuclease which introduces a single-strand nick into specific origin sequences, becoming covalently attached to the 5' end of the DNA at the nick and providing a 3' hydroxyl to prime unidirectional, leading-strand synthesis. Parvoviruses, such as minute virus of mice (MVM), have adapted this mechanism to amplify their linear single-stranded genomes by using hairpin telomeres which sequentially unfold and refold to shuttle the replication fork back and forth along the genome, creating a continuous, multimeric DNA strand. The viral initiator protein, NS1, then excises individual genomes from this continuum by nicking and reinitiating synthesis at specific origins present within the hairpin sequences. Using in vitro assays to study ATP-dependent initiation within the right-hand (5') MVM hairpin, we have characterized a HeLa cell factor which is absolutely required to allow NS1 to nick this origin. Unlike parvovirus initiation factor (PIF), the cellular complex which activates NS1 endonuclease activity at the left-hand (3') viral origin, the host factor which activates the right-hand hairpin elutes from phosphocellulose in high salt, has a molecular mass of around 25 kDa, and appears to bind preferentially to structured DNA, suggesting that it might be a member of the high-mobility group 1/2 (HMG1/2) protein family. This prediction was confirmed by showing that purified calf thymus HMG1 and recombinant human HMG1 or murine HMG2 could each substitute for the HeLa factor, activating the NS1 endonuclease in an origin-specific nicking reaction.

Repression participates in mammary tissue-specific activation of the caprine β-lactoglobulin promoter

Activation of the β-lactoglobulin (BLG) gene promoter is restricted to overtly differentiated mammary tissue. To understand the mechanism underlying such tissue-specificity, activity of the caprine BLG promoter was analyzed comparatively in cultured mammary HC11 cells and non-mammary HeLa and CV-1 cells. The BLG promoter flanked by the 5′-regulatory sequence below −205 was strongly activated in the cells, regardless of the cell type. In non-mammary HeLa and CV-1 cells, this activation was repressed completely by the upper regulatory sequence. Weak repression was also observed in mammary HC11 cells kept non-confluent. As the mammary HC11 cells grew confluent and maintained the stabilized state, however, repression by the upper regulatory sequence was switched to activation. The repressive upstream flanking sequence was strongly recognized by the binding factors in non-mammary HeLa and CV-1 cells in an in vitro binding assay. Binding intensity and competition strength of the upstream regulatory regions were in a close correlation to their transcriptional repression activities in the cultured cells. The results suggest that the restricted activation of the caprine BLG promoter in differentiated mammary tissue is guaranteed by repression in non-mammary and undifferentiated mammary cells.

Positive Effects of SH2 Domain-containing Tyrosine Phosphatase SHP-1 on Epidermal Growth Factor- and Interferon-γ-stimulated Activation of STAT Transcription Factors in HeLa Cells

SHP-1 (also known as PTP1C, SHPTP-1, SHP, and HCP) is an SH2 domain-containing protein-tyrosine phosphatase. We have stably overexpressed the native form and a catalytically inactive cysteine to serine mutant of the enzyme, SHP-1-(Cys --> Ser), in human cervical carcinoma HeLa cells. Following stimulation of the cells with epidermal growth factor (EGF) and interferon-gamma (INF-gamma), signal transducers and activators of transcription (STAT) activity was analyzed by using two 32P-labeled DNA probes, namely hSIE which is derived from a high affinity mutant form of the serum-inducible element in the c-fos promotor and GAS which resembles the INF-gamma activation site. EGF induced hSIE binding activity only, and the activity was suppressed by approximately 70% when the inactive mutant form of SHP-1 was expressed but was essentially unaffected by expression of the native enzyme. INF-gamma treatment resulted in appearance of both hSIE and GAS binding activities. While expression of the inactive mutant reduced the activities by 30-50%, the native enzyme caused a 20-30% increase. Consistent with effects on STAT activation, altered SHP-1 expression also affected EGF-induced activation of the mitogen-activated protein kinase pathway; expression of SHP-1-(Cys --> Ser) inhibited activity of MEK by approximately 25%, whereas expression of SHP-1 resulted in a approximately 25% increase. Further studies revealed that overexpression of SHP-1 caused decreased tyrosine phosphorylation of the EGF receptor and that EGF induced phosphorylation and recruitment of SHP-1. Together, the data suggest that SHP-1 is positively involved in EGF- and INF-gamma-induced STAT activation in non-hematopoietic HeLa cells and that, in the EGF signaling system, SHP-1 functions at least partly by modulating tyrosine phosphorylation of EGF receptor.

A comparison of the substrate specificity of MAPKAP kinase-2 and MAPKAP kinase-3 and their activation by cytokines and cellular stress

MAPKAP kinase-2 and MAPKAP kinase-3 were both activated in response to cellular stress, interleukin-1 and tumour necrosis factor in KB and HeLa cells, and with identical kinetics. Activation of MAPKAP kinase-3, like MAPKAP kinase-2, was prevented by SB 203580, a specific inhibitor of SAPK-2, the upstream activator of MAPKAP kinase-2. MAPKAP kinase-3 and MAPKAP kinase-2 phosphorylated peptide substrates with similar kinetic constants and phosphorylated the same serine residues in HSP27 at the same relative rates. These results establish that MAPKAP kinase-3 lies ‘downstream’ of SAPK-2 and that it is likely to have overlapping or identical substrates to MAPKAP kinase-2 in vivo.

Hypoxia induces AP-1-regulated genes and AP-1 transcription factor binding in human endothelial and other cell types

Hypoxia results in differential expression of specific genes in certain cell types. In endothelial cells, hypoxia activates several genes that are known to be inducible by transcription factor AP-1, including endothelin-1 and platelet-derived growth factor-B (PDGF-B). In this study we demonstrated that other AP-1-inducible genes are activated by hypoxia in these cells, including collagenase IV and c- jun, and sought to correlate the activation of genes by hypoxia with the activation of transcription factor AP-1. Depending upon the type of cell studied, hypoxic exposure resulted in the induction of AP-1 transcription factor DNA-binding activity with wide variations in levels of binding. The magnitude of activation of transcription factor AP-1 by hypoxia did not always strictly correlate with the level of induction of AP-1-inducible genes. This finding indicates a requirement for additional mechanisms of controlling transcription beyond the simple activation of AP-1 factor DNA-binding activity for the activation of AP-1-inducible genes during hypoxia. Hypoxia has been reported to lower the intracellular redox potential. The effect of redox state changes on AP-1 transcription factor activity and on the activation of AP-1-inducible genes was also studied. PDTC, a potent reducing agent, activated the AP-1 transcription factor in HeLa cells, and also resulted in increased accumulation of c- jun mRNA in these cells. In contrast to PDTC-mediated activation of the AP-1 transcription factor and the subsequent induction of the AP-1-regulated c- jun gene, hypoxic activation of AP-1 transcription factor binding to its cognate DNA sequence did not activate the c- jun gene in HeLa cells, thus documenting distinct differences in signals generated by the reducing intracellular microenvironments created by hypoxia and PDTC. These results demonstrate the induction of AP-1 transcription factor activity by hypoxic environments, but suggest that additional factors or cell-specific signals are involved in the regulation of hypoxia-induced genes.

Regulation of Signaling by Receptor Tyrosine Kinases in HeLa Cells Involves the q-Base

The nutrition factor queuine (q; the q-base) is a modulator of mammalian cell proliferation: it can be inhibitory or stimulatory, depending on the metabolic state of the cell. The mechanism underlying this growth-modulating activity was investigated. It was found that the q-base acts antagonistic to epidermal growth factor (EGF) but synergistic with platelet-derived growth factor (PDGF) in HeLa cells. Binding of either growth factor to its receptor resulted in the activation of distinct cellular kinases. The activities of these kinases were profoundly affected by q. The results suggest that the q-base is involved in the homologous regulation of signalling by receptor tyrosine kinases.

Characterization of a HeLa Cell Factor Which Negatively Regulates Transcriptional Activation in Vitro by Transcriptional Enhancer Factor-1 (TEF-1)

A novel negatively acting factor has been identified and partially purified from HeLa and BJA-B cell extracts by chromatographic fractionation. Addition of this factor to HeLa cell extracts or to a reconstituted HeLa cell transcription system repressed transcriptional activation by a chimeric activator, GAL-TEF-1, containing the activation function of transcriptional enhancer factor-1 (TEF-1). In contrast, this factor did not repress transactivation by the chimeric GAL-VP16 activator. Repression of transactivation by GAL-TEF-1 could be alleviated by the addition of immunopurified HeLa cell TFIID, but not by increased quantities of GAL-TEF-1. These observations suggest that this negatively acting factor represses transactivation by interfering with the function of, or competing for, the TATA-binding protein-associated coactivators which mediate the activity TEF-1.

A silencer element for the lipoprotein lipase gene promoter and cognate double- and single-stranded DNA-binding proteins.

Transfection experiments with constructs containing various 5'-deleted fragments of the human lipoprotein lipase (LPL) promoter and the chloramphenicol acetyltransferase reporter gene revealed an LPL silencer element (LSE) in the region of nucleotides -225 to -81 of the LPL gene that functioned in Chinese hamster ovary (CHO) and HeLa cells. Gel retardation competition analysis showed the presence of a nuclear factor(s) capable of binding to the sequence of nucleotides -169 to -152 of LSE (LSE-6) in a single-stranded (opposite-strand) and double-stranded specific fashion, the binding affinity being almost the same in the two binding forms. Site-directed mutagenesis indicated that almost the entire sequence of LSE-6 was necessary to form the complexes and also critical for silencing activity in CHO cells. The amounts of this binding factor(s) in CHO and HeLa cells were closely associated with transcriptional silencing activity. Photochemical cross-linking experiments indicated that the single- and double-stranded elements recognized the same binding factor(s) with molecular masses of 54 to 63 kDa and 109 to 124 kDa. The 109- to 124-kDa DNA binding factor(s) was found to be a doublet of that of the 54- to 63-kDa factor by isoelectric focusing or by increasing the time of exposure to UV irradiation. When inserted upstream of another gene such as that of the simian virus 40 enhancer/promoter of pSV2CAT, the sequence of nucleotides -190 to -143 (LSE-1) also suppressed transcription of the reporter gene in CHO cells. These results strongly suggest that the LSE plays a role in regulation of LPL gene expression by suppressing its transcription.

Temperature-sensitive mutations in the gene encoding the small subunit of the vaccinia virus early transcription factor impair promoter binding, transcription activation, and packaging of multiple virion components.

The vaccinia virus D6R open reading frame encodes the small subunit of the heterodimeric vaccinia virus early transcription factor (VETF) that activates transcription of early genes in vitro. VETF binds early gene promoters and has a DNA-dependent ATPase activity that is essential for activation of transcription. To examine the relationship between the structure and function of VETF, we have localized the mutations in two temperature-sensitive viruses whose lesions previously were mapped to the D6R gene. For both mutants, a single G-to-A nucleotide change that would alter protein coding potential was identified. In mutant E93, the codon for alanine 25 was changed to that of threonine, and in mutant S4 the codon for valine 278 was replaced with that for methionine. The molecular phenotype of each mutant was assessed by expressing mutant transcription factors in HeLa cells by using a vaccinia virus-T7 system and characterizing the proteins' activities in vitro. The A25T mutant activated transcription to a lesser extent than wild-type VETF, and the V278M mutant had no demonstrable transcription factor activity. Both mutant proteins were shown to be defective for promoter binding, accounting for their impairment in transcription activation. The functional defects for both mutants were observed at permissive as well as nonpermissive temperatures. The mutant proteins retained ATPase activity but required higher DNA concentrations to activate the ATPase. These results indicate that the small subunit of VETF is essential for its promoter binding activity and likely contacts the promoter DNA. Immunoblotting experiments showed that the virion particles from the two mutant viruses contained about half the VETF of wild-type virus, suggesting that promoter binding may contribute to packaging of VETF into the virion particle. RNA polymerase, mRNA capping enzyme, and nucleoside triphosphate phosphohydrolase I were found at similarly reduced levels in the virion, indicating that packaging of some virion core enzymes may be interdependent.

Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors: a potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis.

Mitochondrial transcription factor A (mtTFA), the product of a nuclear gene, stimulates transcription from the two divergent mitochondrial promoters and is likely the principal activator of mitochondrial gene expression in vertebrates. Here we establish that the proximal promoter of the human mtTFA gene is highly dependent upon recognition sites for the nuclear respiratory factors, NRF-1 and NRF-2, for activity. These factors have been previously implicated in the activation of numerous nuclear genes that contribute to mitochondrial respiratory function. The affinity-purified factors from HeLa cells specifically bind to the mtTFA NRF-1 and NRF-2 sites through guanine nucleotide contacts that are characteristic for each site. Mutations in these contacts eliminate NRF-1 and NRF-2 binding and also dramatically reduce promoter activity in transfected cells. Although both factors contribute, NRF-1 binding appears to be the major determinant of promoter function. This dependence on NRF-1 activation is confirmed by in vitro transcription using highly purified recombinant proteins that display the same binding specificities as the HeLa cell factors. The activation of the mtTFA promoter by both NRF-1 and NRF-2 therefore provides a link between the expression of nuclear and mitochondrial genes and suggests a mechanism for their coordinate regulation during organelle biogenesis.

Studies on class III gene transcription.

In an attempt to understand the functional role of individual transcription factors, we have carried out S1 nuclease analyses of in vitro synthesized RNA using partially purified HeLa cell factors and truncated 5S and tDNA. The results indicate that faithful initiation of 5S gene transcription depends on the presence of IIIA, IIIB and IIIC in addition to RNA polymerase III. On the other hand, tDNA transcription initiation requires IIIB and IIIC and RNA polymerase III. In addition, we found that either 'A' block promoter or 'B' block alone can not serve as a template for the initiation of tDNA transcription. When an ultraviolet irradiated 5S DNA was used as a template, transcription is minimally affected at a lower dose suggesting that small differences in the helical structure may not have much effect on the transcription machinery. These results along with an in vitro attempt to reproduce Xenopus 5S RNA gene regulation are presented.

An unusual cellular factor potentiates protein-DNA complex assembly between Oct-1 and Vmw65.

The herpes simplex virus trans-activator Vmw65 interacts with the cellular factors Oct-1 and VCAF-1 to generate a multicomponent DNA binding complex that specifically recognizes conserved enhancer elements found upstream of the viral immediate-early genes, resulting in potent stimulation of their transcription. We have identified a HeLa cell factor, distinct from Oct-1 or VCAF-1, which significantly enhances the stability or formation of Vmw65-dependent complexes, as judged by mobility shift analysis using either nuclear extracts or bacterially expressed Oct-1 and Vmw65. This factor, designated SF (stimulatory factor), was partially purified from HeLa cell postnuclear extracts and has an apparent molecular weight of 1500-3000, based on ultrafiltration and size-exclusion chromatography. SF was shown to be resistant to inactivation by heat treatment, protease, nuclease, and phospholipase digestions, and extraction with organic solvents. Pretreatment of SF with beta-glucuronidase did not affect its ability to stimulate Vmw65-dependent complex formation but did reduce the electrophoretic mobility of the resulting complex. These data indicate that SF is probably a component of the Vmw65-induced complex and may be composed, at least partially, of carbohydrate.

A tissue-specific small nuclear ribonucleoprotein and the regulated splicing of the calcitonin/calcitonin gene-related protein transcript.

Based on a correlation between expression patterns of an abundant tissue-specific small nuclear ribonucleoprotein (N protein) and the calcitonin gene-related protein (CGRP) splicing choice, a small nuclear ribonucleoprotein (the N factor) has been hypothesized to potentially function as a trans-acting factor involved in the regulation of the alternative splicing of the calcitonin/CGRP transcript. RNA analysis indicated that most rat, human, and simian cell lines and tissues making the CGRP mRNA splicing choice expressed the N factor mRNA. These data led us to address the effect of ectopic expression of the N factor in HeLa cells, which exhibit a calcitonin splicing choice when expressing the calcitonin/CGRP gene. Expression of the N factor exerts no effect on the calcitonin/CGRP splicing choice in HeLa cells. Furthermore, several cell lines such as the human 293 cell line make the CGRP mRNA splicing choice in the absence of any detectable level of the mRNA encoding the N factor. Together, these data reveal that the N protein is neither sufficient nor required for the tissue-specific CGRP splicing decision and that the N protein is not the trans-acting factor regulating the alternative splicing of the calcitonin/CGRP gene.

Initiation of translation of human rhinovirus RNA: Mapping the internal ribosome entry site

In order to map the 3′ boundary of the segments needed for translation initiation at the correct site on human rhinovirus 2, deletions were made from the 3′ end of the viral 5′-untranslated region. These truncated viral segments were placed immediately upstream of a reporter gene, a derivative of the influenza virus NS cDNA, either as monocistronic constructs or as dicistronic constructs in which the upstream cistron was the Xenopus laevis cyclin B2 cDNA. In vitro transcripts of these clones were translated in the rabbit reticulocyte lysate system, with or without supplementation with crude HeLa cell initiation factors, or in a HeLa cell-free system. When the full-length viral 5′-untranslated region was present, the HeLa cell factors strongly stimulated the synthesis of the NS-related polypeptides, especially in the case of the dicistronic mRNAs. Deletions from the 3′ end extending up to nt 562 had little effect on translation efficiency or the response to HeLa cell factors, but more extensive deletions resulted in the complete loss of response to these factors, an almost total inhibition of NS synthesis from dicistronic mRNAs, and a partial inhibition in the case of the monocistronic mRNAs. In the case of a deletion extending to nt 554, insertion of a 15 nucleotide residue linker failed to restore efficient translation initiation. We conclude that the essential sequences for internal initiation extend to a point located between nt 554 and 562, and that the ribosome entry site, defined as the most 5′-proximal point where the ribosome can bind in an initiation-competent manner, must lie within 6 residues on either side of nt 562, and certainly not further downstream than nt 568.