Untranslated Region RNA Research Articles

Two Yeast PUF Proteins Negatively Regulate a Single mRNA

mRNA stability and translation are regulated by protein repressors that bind 3'-untranslated regions. PUF proteins provide a paradigm for these regulatory molecules: like other repressors, they inhibit translation, enhance mRNA decay, and promote poly(A) removal. Here we show that a single mRNA in Saccharomyces cerevisiae, encoding the HO endonuclease, is regulated by two distinct PUF proteins, Puf4p and Mpt5p. These proteins bind to adjacent sites and can co-occupy the mRNA. Both proteins are required for full repression and deadenylation in vivo; their removal dramatically stabilizes the mRNA. The two proteins act through overlapping but non-identical mechanisms: repression by Puf4p is dependent on deadenylation, whereas repression by Mpt5p can occur through additional mechanisms. Combinatorial action of the two regulatory proteins may allow responses to specific environmental cues and be common in 3'-untranslated region-mediated control.

An RNA G-quadruplex in the 5′ UTR of the NRAS proto-oncogene modulates translation

Guanine-rich nucleic acid sequences can adopt noncanonical four-stranded secondary structures called guanine (G)-quadruplexes. Bioinformatics analysis suggests that G-quadruplex motifs are prevalent in genomes, which raises the need to elucidate their function. There is now evidence for the existence of DNA G-quadruplexes at telomeres with associated biological function. A recent hypothesis supports the notion that gene promoter elements contain DNA G-quadruplex motifs that control gene expression at the transcriptional level. We discovered a highly conserved, thermodynamically stable RNA G-quadruplex in the 5' untranslated region (UTR) of the gene transcript of the human NRAS proto-oncogene. Using a cell-free translation system coupled to a reporter gene assay, we have demonstrated that this NRAS RNA G-quadruplex modulates translation. This is the first example of translational repression by an RNA G-quadruplex. Bioinformatics analysis has revealed 2,922 other 5' UTR RNA G-quadruplex elements in the human genome. We propose that RNA G-quadruplexes in the 5' UTR modulate gene expression at the translational level.

An Apical GAGA Loop within 5’ UTR of the Coxsackievirus B3 RNA Maintains Structural Organization of the IRES Element Required for Efficient Ribosome Entry

Coxsackievirus B3 (CVB3) a close relative of Poliovirus has been implicated as the primary cause of dilated cardiomyopathy in humans. The single-stranded, plus sense, viral genomic RNA is naturally uncapped and the translation of viral encoded protein is initiated from an ‘Internal Ribosome entry site’ (IRES) element at the 5' untranslated region (UTR). The 741 nucleotide 5'UTR is highly structured, with defined stem loops. Previously, we have reported results of probing the secondary structure of the 5'UTR RNA, and showed the existence of 11 putative stem loop (SL) structures, SL A-K. Further, using mutants, we showed the importance of an apical GAGA loop in SL-H, in IRES activity. In addition to significantly reducing CVB3 IRES activity the mutations in the 5'UTR drastically reduced interaction with La autoantigen, a critical trans acting factor. Here, we demonstrate that mutation of the GAGA loop to CAGU alters the structure around SL-H, and reduces the interaction of La protein with critical cis acting elements of the IRES RNA. Interestingly, mutation of the loop to GAAA also has similar effect, suggesting the GAGA sequence of the loop, is necessary for the function. Furthermore, using gene specific siRNA against La protein we have demonstrated La protein to be a bona fide trans acting factor for CVB3 IRES. However, upon partial knockdown of La protein, translation mediated by the mutant 5'UTRs are affected to a similar extent as the wild type, implicating the structural alteration as the reason for the effect of the mutations on IRES activity.

Molecular Mechanisms of Germ Line Cell Determination in Animals

The principles of the formation of germ line cells are reviewed. Germ line cells are separated from other embryonic cells during early embryogenesis. In some groups of animals, germ cell precursors are induced by surrounding cells. In most taxa, however, cells that have inherited certain maternal determinants (the so-called germ plasm) become primordial germ cells. Germ plasm usually contains mitochondria, electron-dense granules with complex structure, and maternal proteins and RNAs necessary to form germ line cells. In Xenopus, the mitochondrial cloud is the source of material for germ plasm; it also specifically binds maternal RNAs involved in the formation of primordial germ cells and transports them to the vegetal pole. The cis-acting elements that are responsible for the transport of these RNAs are usually located in the 3′-untranslated region of RNA, and their function is mediated by binding with trans-acting protein factors. In addition to specific localization of macromolecules in germ plasm, the special status of germ line cells is ensured by degradation of RNA and the protein components of germ plasm in somatic cells, total suppression of transcription in primordial germ line cells until advanced stages of embryogenesis, and specific regulation of RNA translation in somatic and germ line cells. Experimental data on the characteristics of the germes maternal RNA, which is a novel component of the Xenopus germ plasm, and the protein that it encodes are briefly discussed.

The pathogenic agent in Drosophila models of ‘polyglutamine’ diseases

A substantial body of evidence supports the identity of polyglutamine as the pathogenic agent in a variety of human neurodegenerative disorders where the mutation is an expanded CAG repeat. However, in apparent contradiction to this, there are several human neurodegenerative diseases (some of which are clinically indistinguishable from the 'polyglutamine' diseases) that are due to expanded repeats that cannot encode polyglutamine. As polyglutamine cannot be the pathogenic agent in these diseases, either the different disorders have distinct pathogenic pathways or some other common agent is toxic in all of the expanded repeat diseases. Recently, evidence has been presented in support of RNA as the pathogenic agent in Fragile X-associated tremor/ataxia syndrome (FXTAS), caused by expanded CGG repeats at the FRAXA locus. A Drosophila model of FXTAS, in which 90 copies of the CGG repeat are expressed in an untranslated region of RNA, exhibits both neurodegeneration and similar molecular pathology to the 'polyglutamine' diseases. We have, therefore, explored the identity of the pathogenic agent, and specifically the role of RNA, in a Drosophila model of the polyglutamine diseases by the expression of various repeat constructs. These include expanded CAA and CAG repeats and an untranslated CAG repeat. Our data support the identity of polyglutamine as the pathogenic agent in the Drosophila models of expanded CAG repeat neurodegenerative diseases.

Translational Control of β2-Adrenergic Receptor mRNA by T-cell-restricted Intracellular Antigen-related Protein

Cellular expression of the beta(2)-adrenergic receptor (beta(2)-AR) is suppressed at the translational level by 3'-untranslated region (UTR) sequences. To test the possible role of 3'-UTR-binding proteins in translational suppression of beta(2)-AR mRNA, we expressed the full-length 3'-UTR or the adenylate/uridylate-rich (A+U-rich element (ARE)) RNA from the 3'-UTR sequences of beta(2)-AR in cell lines that endogenously express this receptor. Reversal of beta(2)-adrenergic receptor translational repression by retroviral expression of 3'-UTR sequences suggested that ARE RNA-binding proteins are involved in translational suppression of beta(2)-adrenergic receptor expression. Using a 20-nucleotide ARE RNA from the receptor 3'-UTR as an affinity ligand, we purified the proteins that bind to these sequences. T-cell-restricted intracellular antigen-related protein (TIAR) was one of the strongly bound proteins identified by this method. UV-catalyzed cross-linking experiments using in vitro transcribed 3'-UTR RNA and glutathione S-transferase-TIAR demonstrated multiple binding sites for this protein on beta(2)-AR 3'-UTR sequences. The distal 340-nucleotide region of the 3'-UTR was identified as a target RNA motif for TIAR binding by both RNA gel shift analysis and immunoprecipitation experiments. Overexpression of TIAR resulted in suppression of receptor protein synthesis and a significant shift in endogenously expressed beta(2)-AR mRNA toward low molecular weight fractions in sucrose gradient polysome fractionation. Taken together, our results provide the first evidence for translational control of beta(2)-AR mRNA by TIAR.

Isolation and identification of a protein binding to the localization element of Metallothionein-1 mRNA

mRNA localization provides a mechanism for localized protein synthesis. mRNAs encoding certain proteins, including c-MYC, c-FOS, MT-1 (Metallothionein-1) and vimentin, are localized around the nuclei of mammalian cells and are associated with the cytoskeleton. Targeting of these mRNAs to the perinuclear cytoplasm is mediated by elements within their 3'-UTRs (3'-untranslated regions), but many of the trans-acting proteins remain unidentified. UV cross-linking assays using radiolabelled transcripts indicated that a protein of approx. 50 kDa (from the Chinese-hamster ovary cell extracts) bound to the MT-1 3'-UTR sequence. Competition experiments using unlabelled mutant 3'-UTR RNAs revealed that the binding of this protein is specific to localization-positive mutants. Isolation of a 50 kDa protein was achieved by an RNA affinity-based method in which biotinylated MT-1 3'-UTR RNA was anchored to paramagnetic beads. Bound proteins were eluted and analysed by SDS/PAGE. The 50 kDa protein was extracted from the gel, subjected to trypsin digestion and identified by matrix-assisted laser-desorption/ionization-time-of-flight mass spectrometry as eukaryote elongation factor 1alpha.

Specific Interaction between Human Parechovirus Nonstructural 2A Protein and Viral RNA

The functional properties of the nonstructural 2A protein are variable among different picornaviruses. The 2A protein of the human parechovirus 1 (HPEV1) has been shown to lack the proteolytic activity found in many other picornaviruses, but no particular function has been identified for HPEV1 2A. To obtain information about the role of HPEV1 2A in the viral life cycle, the protein was expressed in Escherichia coli. A polyclonal antibody was then raised against the protein and employed to investigate its subcellular localization in the infected cells by immunofluorescence microscopy. Typically, a diffuse cytoplasmic staining pattern, concentrated to the perinuclear area, was observed in the infected cells. However, at late stages of infection some infected cells also exhibited diffuse nuclear staining. Viral RNA, visualized by fluorescent in situ hybridization, partly colocalized with 2A in the perinuclear region. Three experimental approaches including Northwestern blot, UV cross-linking, and gel retardation demonstrated that 2A possesses RNA binding activity. Competition experiments with various single-stranded RNA molecules addressed the specificity of 2A binding. These studies revealed that the 2A protein bound RNA corresponding to the 3'-untranslated region (UTR) of the viral genome with highest affinity. At the N- and C-terminal ends of the protein, two regions, necessary for RNA binding, were identified by mutagenesis. In addition, we demonstrated that 2A has affinity to double-stranded RNA containing 3'UTR(+)-3'UTR(-). In conclusion, our experiments showed that HPEV1 2A binds to viral 3'UTR RNA, a feature that could be important for the function of the protein during HPEV1 replication.

Cellular proteins from human monocytes bind to dengue 4 virus minus-strand 3' untranslated region RNA.

The synthesis of plus and minus RNA strands of several RNA viruses requires as a first step the interaction of some viral regulatory sequences with cellular and viral proteins. The dengue 4 virus genome, a single-stranded, positive-polarity RNA, is flanked by two untranslated regions (UTR) located in the 5' and 3' ends. The 3'UTR in the minus-strand RNA [3'UTR (-)] has been thought to function as a promoter for the synthesis of plus-strand RNA. To study the initial interaction between this 3'UTR and cellular and viral proteins, mobility shift assays were performed, and four ribonucleoprotein complexes (I through IV) were formed when uninfected and infected U937 cells (human monocyte cell line) interacted with the 3'UTR (-) of dengue 4 virus. Cross-linking assays with RNAs containing the complete 3'UTR (-) (nucleotides [nt] 101 to 1) or a partial sequence from nt 101 to 45 and nt 44 to 1 resulted in specific binding of some cellular proteins. Supermobility shift and immunoprecipitation assays demonstrated that the La protein forms part of these complexes. To determine the region in the 3' UTR that interacted with the La protein, two deletion mutants were generated. The mutant (del-96), with a deletion of nt 96 to 101, was unable to interact with the La protein, suggesting that La interacted with the 5' portion of the 3'UTR (-). Complex I, which was the main ribonucleoprotein complex formed with the 3'UTR (-) and which had the fastest electrophoretic migration, contained proteins such as calreticulin and protein disulfide isomerase, which constitute important components of the endoplasmic reticulum.

Sarcoplasmic reticulum Ca(2+) pump mRNA stability in cardiac and smooth muscle: role of the 3'-untranslated region.

Stomach smooth muscle (SSM) and left ventricular muscle (LVM) express the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump gene SERCA2. Alternative splicing yields two major isoforms, SERCA2a in LVM and slow twitch muscle and SERCA2b in SSM and most other tissues. The splices have different 3'-untranslated regions (UTR) and also encode proteins that differ slightly in their COOH-terminal domains. SERCA2 transcription rates are similar in the two tissues, yet LVM has a much higher level of SERCA2 mRNA than SSM. To understand the control of SERCA2 RNA expression, we inhibited transcription and showed that the half-life of SERCA2 mRNA is significantly longer (P < 0.05) in primary cultures of LVM cells than in SSM cells. Nuclear SERCA2 mRNA levels were also higher in LVM than in SSM. In vitro decay assays using synthetic RNA corresponding to the 3'-UTR of SERCA2a and -2b showed that nuclear extracts produced a faster decay of SERCA2 RNA than cytoplasmic extracts and that nuclear extracts produced a faster decay of SERCA2b than -2a. This was also true when the full-length native mRNA was used instead of the 3'-UTR RNA, and SERCA2b decay by cytoplasmic extracts was faster for LVM than for SSM. We propose that nuclear decay is an initial step in the control of SERCA2 RNA abundance and that this control is maintained or modulated in the cytoplasm. We discuss how these control mechanisms may be part of a control switch in cardiac development and pathophysiology.

Internal initiation sites of de novo RNA synthesis within the hepatitis C virus polypyrimidine tract

A variety of 3 ′-untranslated regions (UTRs) were cloned from infectious hepatitis C virus human samples and examined in NS5B polymerase de novo initiation reactions. We isolated and characterized four distinct 3 ′-UTRs that harbor the conserved terminal 98 nucleotides, but have poly(U/UC) tracts of 25, 93, 98, and 101 nucleotides, respectively. Reconstitution of de novo initiation by the mature NS5B with the different 3 ′-UTR RNA substrates revealed distinctively sized products that are consistent with internal initiation at specific sites within the polypyrimidine tract. These sites were further mapped by demonstrating that nucleotide substitutions of the cytidylate stretches within the poly(U/UC) template eliminate specific products of de novo synthesis and by showing that these products could be radiolabeled by [γ- 32P]GTP. We also examine analogs that can substitute for GTP in this reaction.

Binding of the La autoantigen to the 5′ untranslated region of a chimeric human translation elongation factor 1A reporter mRNA inhibits translation in vitro

Human translation elongation factor 1A (EF1A) is a member of a large class of mRNAs, including ribosomal proteins and other translation elongation factors, which are coordinately translationally regulated under various conditions. Each of these mRNAs contains a t erminal o ligo p yrimidine tract (TOP) that is required for translational control. A human growth hormone (hGH) expression construct containing the promoter region and 5′ untranslated region (UTR) of EF1A linked to the hGH coding region (EF1A/hGH) was translationally repressed following rapamycin treatment in similar fashion to endogenous EF1A in human B lymphocytes. Mutation of two nucleotides in the TOP motif abolished the translational regulation. Gel mobility shift assays showed that both La protein from human B lymphocyte cytoplasmic extracts as well as purified recombinant La protein specifically bind to an in vitro-synthesized RNA containing the 5′ UTR of EF1A mRNA. Moreover, extracts prepared from rapamycin-treated cells showed increased binding activity to the EF1A 5′ UTR RNA, which correlates with TOP mRNA translational repression. In an in vitro translation system, recombinant La dramatically decreased the expression of EF1A/hGH construct mRNA, but not mRNAs lacking an intact TOP element. These results indicate that TOP mRNA translation may be modulated through La binding to the TOP element.

Role of the 3'-untranslated regions of alfalfa mosaic virus RNAs in the formation of a transiently expressed replicase in plants and in the assembly of virions.

Alfalfa mosaic virus (AMV) RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and the coat protein (CP). When RNAs 1 and 2 were transiently expressed from a T-DNA vector (R12 construct) by agroinfiltration of Nicotiana benthamiana, the infiltrated leaves accumulated minus-strand RNAs 1 and 2 and relatively small amounts of plus-strand RNAs. In addition, RNA-dependent RNA polymerase (RdRp) activity could be detected in extracts of the infiltrated leaves. After transient expression of RNAs 1 and 2 with the 3'-untranslated regions (UTRs) of both RNAs deleted (R1Delta/2Delta construct), no replication of RNAs 1 and 2 was observed, while the infiltrated leaves supported replication of RNA 3 after inoculation of the leaves with RNA 3 or expression of RNA 3 from a T-DNA vector (R3 construct). No RdRp activity could be isolated from leaves infiltrated with the R1Delta/2Delta construct, although P1 and P2 sedimented in a region of a glycerol gradient where active RdRp was found in plants infiltrated with R12. RdRp activity could be isolated from leaves infiltrated with constructs R1Delta/2 (3'-UTR of RNA 1 deleted), R1/2Delta (3'-UTR of RNA 2 deleted), or R1Delta/2Delta plus R3. This demonstrates that the 3'-UTR of AMV RNAs is required for the formation of a complex with in vitro enzyme activity. RNAs 1 and 2 with the 3'-UTRs deleted were encapsidated into virions by CP expressed from RNA 3. This shows that the high-affinity binding site for CP at the 3'-termini of AMV RNAs is not required for assembly of virus particles.

Down-regulation of translation driven by hepatitis C virus internal ribosomal entry site by the 3' untranslated region of RNA.

The genome of hepatitis C virus (HCV) is a single-stranded RNA of positive polarity that has a poly(U/C) tract followed by a highly conserved 98-nt sequence, termed the X region, in the 3' untranslated region (UTR). To investigate the effect of the 3'UTR on the HCV translation that depends on the internal ribosomal entry site (IRES), we prepared a deletion HCV RNA, MA delta, that lacked the RNA region from nt 1286 to 8785. A series of MA delta RNAs that differ in the primary structure of their 3'UTR, were generated and examined for their translation efficiencies in reticulocyte lysates. Deletion of the poly(U/C) tract and/or stem-loop structure (SL) 3 region of 3'X resulted in enhancement of the translation efficiency. Translation of MA delta RNA was inhibited by the addition of recombinant polypyrimidine tract-binding protein (PTB). A similar inhibition by PTB, however, was observed when an RNA lacking the poly(U/C) tract or SL3 region was used. The inhibitory effect by PTB was not obvious for MA delta (1041) RNA composed of nt 1 to 1041 but MA delta (8928) RNA composed of nt 1 to 1285 and nt 8786 to 8928. These results suggest that the observed down-regulation of HCV translation by the 3'UTR is mediated by some host factor(s) other than PTB, and that a PTB site for inhibition resides in the coding sequence of nt 1042 to 8928 of MA delta RNA.

An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast.

The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

Human CKIα L and CKIα S are encoded by both 2.4- and 4.2-kb transcripts, the longer containing multiple RNA-destablising elements

Casein kinase I (CKI) are a family of conserved second messenger-independent serine/threonine protein kinases found in all eukaryotes. The avian and mammalian CKI alpha isoform has four splice variants differing in the presence or absence of 28 amino acids (‘ L’ insertion) in the catalytic domain and/or 12 amino acids (‘ S’ insertion) in the regulatory domain. Here we report the isolation of cDNAs encoding human CKIα L and CKIα S. We find human CKIα L has a preference to phosphorylate phosvitin over casein, with a higher K m for casein than phosvitin, the reverse being the case for human CKIα S. Both human CKIα L, and CKIα S are derived from 4.2-kb mRNA transcripts and 2.4-kb transcripts, the latter probably generated by use of an alternate polyadenylation signal identified in the longer transcripts. The 4.2-kb transcripts contain six RNA-destabilising AU-rich element (ARE) motifs in the 3′-untranslated region (UTR), while the 2.4-kb transcripts contain a single ARE motif. In vitro analysis of CKI alpha 3′-UTR RNA sequences suggests that in HeLa cells, the longer 3′-UTR transcripts are likely to degrade approximately 13 times faster than the shorter 3′-UTR transcripts. This is the first report of a kinase mRNA containing multiple RNA-destabilising AREs in the longer of two mRNA transcripts.

A single-stranded loop in the 5′ untranslated region of cucumber mosaic virus RNA 4 contributes to competitive translational activity

The 5′ untranslated region (UTR) of cucumber mosaic virus (CMV) RNA 4 confers a highly competitive translational advantage on a heterologous luciferase open reading frame. Here we investigated whether secondary structure in the 5′ UTR contributes to this translational advantage. Stabilization of the 5′ UTR RNA secondary structure inhibited competitive translational activity. Alteration of a potential single-stranded loop to a stem by substitution mutations greatly inhibited the competitive translational activity. Tobacco plants infected with wild type virus showed a 2.5-fold higher accumulation of maximal coat protein than did plants infected with a loop-mutant virus. Amplification of viral RNA in these plants could not explain the difference in accumulation of coat protein. Phylogenetic comparison showed that potential single-stranded loops of 12–23 nucleotides in length exist widely in subgroups of CMV.

A Novel in Vitro Replication System for Dengue Virus: INITIATION OF RNA SYNTHESIS AT THE 3′-END OF EXOGENOUS VIRAL RNA TEMPLATES REQUIRES 5′- AND 3′-TERMINAL COMPLEMENTARY SEQUENCE MOTIFS OF THE VIRAL RNA

Positive strand viral replicases are membrane-bound complexes of viral and host proteins. The mechanism of viral replication and the role of host proteins are not well understood. To understand this mechanism, a viral replicase assay that utilizes extracts from dengue virus-infected mosquito (C6/36) cells and exogenous viral RNA templates is reported in this study. The 5'- and 3'-terminal regions (TR) of the template RNAs contain the conserved elements including the complementary (cyclization) motifs and stem-loop structures. RNA synthesis in vitro requires both 5'- and 3'-TR present in the same template molecule or when the 5'-TR RNA was added in trans to the 3'-untranslated region (UTR) RNA. However, the 3'-UTR RNA alone is not active. RNA synthesis occurs by elongation of the 3'-end of the template RNA to yield predominantly a double-stranded hairpin-like RNA product, twice the size of the template RNA. These results suggest that an interaction between 5'- and 3'-TR of the viral RNA that modulates the 3'-UTR RNA structure is required for RNA synthesis by the viral replicase. The complementary cyclization motifs of the viral genome also seem to play an important role in this interaction.

Complete sequence of RNA 1 and the presence of tRNA-like structures in all RNAs of Potato mop-top virus, genus Pomovirus.

The complete nucleotide sequence (6043 nt) of RNA 1 from Potato mop-top virus (PMTV-Sw), the type member of the genus Pomovirus, was determined. The first (5'-terminal) open reading frame (ORF 1) encodes a predicted protein of 148 kDa. ORF 2 extends through the opal stop codon of ORF 1 producing a predicted readthrough protein of 206 kDa which resembles the RNA-dependent RNA polymerases (RdRp) of other fungal-transmitted viruses. It includes a methyltransferase, a helicase and a GDD RdRp motif, respectively. Phylogenetic analyses of RdRps indicated that PMTV is most closely related to Beet soil-borne virus (genus Pomovirus), Broad bean necrosis virus (genus Pomovirus) and Soil-borne wheat mosaic virus (genus Furovirus), and is more distantly related to the other viruses of the former furovirus group. The 5' and 3' termini of RNA 1 in PMTV contained untranslated regions (UTR) of 114 nt and 489 nt, respectively. The 3'-UTR of RNA 1 contained a tRNA-like structure, which has previously been reported in the 3'-UTR of RNA 2 but not RNA 3. However, in this study, the tRNA-like structure was also found in the 3'-UTR of RNA 3, which confirms its presence in the 3'-UTRs of all three RNAs of PMTV.

Identification of cis and trans elements involved in the cell cycle regulation of multiple genes in Crithidia fasciculata.

Transcripts of several DNA replication genes, including the RPA1 and TOP2 genes, encoding the large subunit of nuclear replication protein A and the kinetoplast topoisomerase II, accumulate periodically during the cell cycle in the trypanosomatid Crithidia fasciculata. An octamer consensus sequence, CAUAGAAG, present in the 5' untranslated regions (UTR) of these mRNAs is required for periodic accumulation of the TOP2 and RPA1 transcripts and also for binding of a nuclear factor(s) to the 5' UTR RNAs of these genes. We show here that insertion of multiple (six) copies of this octamer sequence (6x octamer) into the 5' UTR of a reporter gene confers periodic accumulation on its transcript. Competition experiments and UV cross-linking studies show that the 6x octamer RNA and TOP2 5' UTR RNA bind to the same nuclear factor(s). Single-nucleotide substitutions in the 6x octamer that abolish the RNA gel shift also prevent cyclic accumulation of the reporter gene transcript. A protein termed cycling element binding protein, purified by affinity chromatography using 6x octamer RNA as a ligand, binds to RNAs containing wild-type octamers and not to those with mutant octamers. These results define a small sequence element in C. fasciculata mRNAs required for their cell cycle regulation and report the identification and purification of a putative regulatory protein that binds specifically to these elements.