Pyrimidine Tract Research Articles

A novel set of nuclear localization signals determine distributions of the alphaCP RNA-binding proteins.

AlphaCPs comprise a subfamily of KH-domain-containing RNA-binding proteins with specificity for C-rich pyrimidine tracts. These proteins play pivotal roles in a broad spectrum of posttranscriptional events. The five major alphaCP isoforms are encoded by four dispersed loci. Each isoform contains three repeats of the RNA-binding KH domain (KH1, KH2, and KH3) but lacks other identifiable motifs. To explore the complexity of their respective functions, we examined the subcellular localization of each alphaCP isoform. Immunofluorescence studies revealed three distinct distributions: alphaCP1 and alphaCP2 are predominantly nuclear with specific enrichment of alphaCP1 in nuclear speckles, alphaCP3 and alphaCP4 are restricted to the cytoplasm, and alphaCP2-KL, an alphaCP2 splice variant, is present at significant levels in both the nucleus and the cytoplasm. We mapped nuclear localization signals (NLSs) for alphaCP isoforms. alphaCP2 contains two functionally independent NLS. Both NLSs appear to be novel and were mapped to a 9-amino-acid segment between KH2 and KH3 (NLS I) and to a 12-amino-acid segment within KH3 (NLS II). NLS I is conserved in alphaCP1, whereas NLS II is inactivated by two amino acid substitutions. Neither NLS is present in alphaCP3 or alphaCP4. Consistent with mapping studies, deletion of NLS I from alphaCP1 blocks its nuclear accumulation, whereas NLS I and NLS II must both be inactivated to block nuclear accumulation of alphaCP2. These data demonstrate an unexpected complexity in the compartmentalization of alphaCP isoforms and identify two novel NLS that play roles in their respective distributions. This complexity of alphaCP distribution is likely to contribute to the diverse functions mediated by this group of abundant RNA-binding proteins.

Differential translation of TOP mRNAs in rapamycin-treated human B lymphocytes

TOP mRNAs (contain a 5′ terminal oligo pyrimidine tract) are differentially translated in rapamycin-treated human B lymphocytes. Following rapamycin treatment, ribosomal protein (rp) and translation elongation factor TOP mRNAs were translationally repressed, whereas hnRNP A1 TOP mRNA was not. Poly(A)-binding protein (Pabp1) TOP mRNA was translationally repressed under all conditions tested. To investigate the mechanism involved, chimeric mRNAs containing the hnRNP A1 5′ untranslated region (UTR) linked to the human growth hormone (hGH) reporter were analyzed. Wild-type hnRNP A1 construct mRNA behaved similarly to endogenous hnRNP A1, whereas a single mutation (guanosine to cytidine) within the TOP element resulted in increased translational regulation. These results suggest that TOP mRNA translation can be modulated and that all TOP mRNAs are not translated with equal efficiency.

Identification of the SRC pyrimidine-binding protein (SPy) as hnRNP K: implications in the regulation of SRC1A transcription.

The human SRC gene encodes pp60(c-src), a non-receptor tyrosine kinase involved in numerous signaling pathways. Activation or overexpression of c-Src has also been linked to a number of important human cancers. Transcription of the SRC gene is complex and regulated by two closely linked but highly dissimilar promoters, each associated with its own distinct non-coding exon. In many tissues SRC expression is regulated by the housekeeping-like SRC1A promoter. In addition to other regulatory elements, three substantial polypurine:polypyrimidine (TC) tracts within this promoter are required for full transcriptional activity. Previously, we described an unusual factor called SRC pyrimidine-binding protein (SPy) that could bind to two of these TC tracts in their double-stranded form, but was also capable of interacting with higher affinity to all three pyrimidine tracts in their single-stranded form. Mutations in the TC tracts, which abolished the ability of SPy to interact with its double-stranded DNA target, significantly reduced SRC1A promoter activity, especially in concert with mutations in critical Sp1 binding sites. Here we expand upon our characterization of this interesting factor and describe the purification of SPy from human SW620 colon cancer cells using a DNA affinity-based approach. Subsequent in-gel tryptic digestion of purified SPy followed by MALDI-TOF mass spectrometric analysis identified SPy as heterogeneous nuclear ribonucleoprotein K (hnRNP K), a known nucleic-acid binding protein implicated in various aspects of gene expression including transcription. These data provide new insights into the double- and single-stranded DNA-binding specificity, as well as functional properties of hnRNP K, and suggest that hnRNP K is a critical component of SRC1A transcriptional processes.

A very compact mt-DNA control region in the widely distributed goby Pomatoschistus minutus (Teleostei: Gobiidae)

The control region of the mitochondrial genome was amplified and sequenced for six individuals of the gobioid fish Pomatoschistus minutus, from several European localities, and one specimen of the related Deltentosteus quadrimaculatus. The length of this region for the former species was found to be 773 bp, 7·1% shorter than that previously described as the most compact D-loop known among teleosts. Sequences from other fish have been compared and the largest gap falls in the section between the conserved sequence block and the pyrimidine tract. Alignment of P. minutus sequences was done with D. quadrimaculatus, whose control region length was 853 bp, and this gap was found to be of 61 bp. For the P. minutus sample, the intraspecific sequence divergence is 0·07%.

Pre-spliceosome formation in S.pombe requires a stable complex of SF1-U2AF(59)-U2AF(23).

We have initiated a biochemical analysis of splicing complexes in extracts from the fission yeast Schizosaccharomyces pombe. Extracts of S.pombe contain high levels of the spliceosome-like U2/5/6 tri-snRNP, which dissociates into mono-snRNPs in the presence of ATP, and supports binding of U2 snRNP to the 3' end of introns, yielding a weak ATP-independent E complex and the stable ATP-dependent complex A. The requirements for S.pombe complex A formation (pre-mRNA sequence elements, protein splicing factors, SF1/BBP and both subunits of U2AF) are analogous to those of mammalian complex A. The S.pombe SF1/BBP, U2AF(59) and U2AF(23) are tightly associated in a novel complex that is required for complex A formation. This pre-formed SF1- U2AF(59)-U2AF(23) complex may represent a streamlined mechanism for recognition of the branch site, pyrimidine tract and 3' splice site at the 3' end of introns.

A novel function for the U2AF 65 splicing factor in promoting pre-mRNA 3'-end processing.

Splicing and 3'-end processing (including cleavage and polyadenylation) of vertebrate pre-mRNAs are tightly coupled events that contribute to the extensive molecular network that coordinates gene expression. Sequences within the terminal intron of genes are essential to stimulate pre-mRNA 3'-end processing, although the factors mediating this effect are unknown. Here, we show that the pyrimidine tract of the last splice acceptor site of the human beta-globin gene is necessary to stimulate mRNA 3'-end formation in vivo and binds the U2AF 65 splicing factor. Naturally occurring beta-thalassaemia-causing mutations within the pyrimidine tract reduces both U2AF 65 binding and 3'-end cleavage efficiency. Significantly, a fusion protein containing U2AF 65, when tethered upstream of a cleavage/polyadenylation site, increases 3'-end cleavage efficiency in vitro and in vivo. Therefore, we propose that U2AF 65 promotes 3'-end processing, which contributes to 3'-terminal exon definition.

Role of PTB-like protein, a neuronal RNA-binding protein, during the differentiation of PC12 cells.

PTB-like protein (PTBLP) is a new homologue of pyrimidine tract binding protein (PTB), and has been cloned as a possible autoantigen in cancer-associated retinopathy. PTBLP has two functional domains, the nuclear localization signal and the RNA recognition motifs (RRMs). Full-length PTBLP (PTBLP-L) has four RRMs, and its alternative splicing product (PTBLP-S) lacks the third and fourth RRMs. Although PTBLPs are expressed in neuronal tissues, the function of PTBLPs has not been determined. We have studed whether PTBLP plays a role in neuronal differentiation using PC12 cells. During the process of nerve growth factor-induced neuronal differentiation of PC12 cells, PTBLP-L was down-regulated whereas PTBLP-S was up-regulated. Transfection of PTBLP-L into PC12 cells led to the suppression of neuronal differentiation. In PTBLP-S transfected cells, however, this suppression was not evident. When both PTBLP-L and PTBLP-S were co-transfected, the suppressive effect of PTBLP-L decreased. In differentiated cells, PTBLP-S localized in the nucleus and PTBLP-L was found dispersed throughout the cytoplasm and neuronal growth cone. These findings suggest that PTBLP-L acts as a negative regulator of neuronal differentiation and PTBLP-S acts as a competitor of PTBLP-L.

Characterization of the sequences encoding for Xenopus laevis box C/D snoRNP Nop56 protein

Nop56p was initially identified in yeast as the third common component of the ribonucleoprotein particles (snoRNPs) assembled on box C/D small nucleolar RNAs (snoRNAs). Thereafter, the characterization of Nop56p homologs in Archaea and in several eukaryotes pointed to the highly conserved structure of this factor. Studies in yeast indicate that Nop56 is not required for the stability of box C/D snoRNAs. Through the isolation of a Xenopus laevis Nop56 cDNA clone, we have been able to characterize the X. laevis Nop56 protein (XNop56p). We showed that it is a common component of X. laevis box C/D snoRNPs and that it displays the same electrophoretic mobility of p62 protein that we previously characterized as a box C/D snoRNP component, not essential for snoRNA stability in X. laevis. Mapping the 5′ end of X. laevis Nop56 transcript indicates that it starts with a pyrimidine tract and the analysis of genomic clones revealed a snoRNA encoded in one of NOP56 introns. Although these two characteristics could suggest that XNOP56 is a TOP gene, it is not translationally controlled in a growth-dependent manner.

Bias of purine stretches in sequenced chromosomes

We examined more than 700 DNA sequences (full length chromosomes and plasmids) for stretches of purines (R) or pyrimidines (Y) and alternating YR stretches; such regions will likely adopt structures which are different from the canonical B-form. Since one turn of the DNA helix is roughly 10 bp, we measured the fraction of each genome which contains purine (or pyrimidine) tracts of lengths of 10 bp or longer (hereafter referred to as ‘purine tracts’), as well as stretches of alternating pyrimidines/purine (‘pyr/pur tracts’) of the same length. Using this criteria, a random sequence would be expected to contain 1.0% of purine tracts and also 1.0% of the alternating pyr/pur tracts. In the vast majority of cases, there are more purine tracts than would be expected from a random sequence, with an average of 3.5%, significantly larger than the expectation value. The fraction of the chromosomes containing pyr/pur tracts was slightly less than expected, with an average of 0.8%. One of the most surprising findings is a clear difference in the length distributions of the regions studied between prokaryotes and eukaryotes. Whereas short-range correlations can explain the length distributions in prokaryotes, in eukaryotes there is an abundance of long stretches of purines or alternating purine/pyrimidine tracts, which cannot be explained in this way; these sequences are likely to play an important role in eukaryotic chromosome organisation.

An improved method for detection and quantification of differential interactions between poliovirus internal ribosome entry site RNA and pyrimidine tract binding protein from primary cells

Attenuated and pathogenic viral variants are often extremely similar viruses with drastically different replication potentials. Despite precise knowledge of viral residues responsible for poliovirus attenuation/neurovirulence, molecular mechanisms mediating these effects remain poorly understood. Data from numerous sources suggest that a functional difference in translation initiation is one responsible factor. However, direct evidence, as well as a comprehensive model are lacking. Several difficulties, including lack of an assay system to quantify differential internal ribosome entry site/pyrimidine tract binding protein interaction in relevant systems, have precluded progress. A novel assay system that overcomes some difficulties is presented below. The assay uses streptavidin paramagnetic particles, biotinylated RNA and glutathione-S-transferase/pyrimidine tract binding protein fusion to detect nanogram levels of uncloned cellular pyrimidine tract binding protein species that interact with internal ribosome entry site RNA. Using this assay, it was shown that pyrimidine tract binding protein from primary human monocytes binds to internal ribosome entry site RNA from virulent poliovirus better than to that from attenuated virus, while pyrimidine tract binding protein from HeLa cells does not distinguish between the two internal ribosome entry sites. Since primary human monocytes reflect neurovirulence-related differential poliovirus replication, these results suggest that pyrimidine tract binding protein may contribute to differential poliovirus replication in vivo. This assay also has the potential to be applicable broadly to other nucleic acid/protein interactions.

The internal ribosome entry site (IRES) of hepatitis C virus visualized by electron microscopy.

Translation of hepatitis C virus (HCV) RNA is initiated via the internal ribosome entry site (IRES), located within the 5' untranslated region. Although the secondary structure of this element has been predicted, little information on the tertiary structure is available. Here we report the first structural characterization of the HCV IRES using electron microscopy. In vitro transcribed RNA appeared as particles with characteristic morphology and gold labeling using a specific oligonucleotide confirmed them to be HCV IRES. Dimerization of the IRES by hybridization with tandem repeat oligonucleotides allowed the identification of domain III and an assignment of domains II and IV to distinct regions within the molecule. Using immunogold labeling, the pyrimidine tract binding protein (PTB) was shown to bind to domain III. Structure-function relationships based on the flexible hinge between domains II and III are suggested. Finally, the architecture of the HCV IRES was seen to be markedly different from that of a picornavirus, foot-and-mouth disease virus (FMDV).

Pyrimidine tract binding protein and La autoantigen interact differently with the 5′ untranslated regions of lentiviruses and oncoretrovirus mRNAs

Retrovirus genomic mRNA exhibits a several hundred nucleotides-long untranslated region (5′ UTR) which encloses many control elements required for retrovirus replication. In addition, this 5′ UTR contains translation regulatory elements, such as internal ribosome entry sites (IRESes) that have been described in oncoretroviruses, as well as in lentiviruses. UV cross-linking experiments suggested that the pyrimidine tract binding protein (PTB), a cellular protein known to regulate the activity of several picornaviral IRESes, binds to human T-cell leukemia virus (HTLV)-I RNA but not to lentiviral human immunodeficiency virus (HIV)-1, HIV-2 or simian immunodeficiency virus RNAs. To calculate the affinity of such RNA–protein interactions, we developed a new method based on the BIAcore technology. The absence of affinity of PTB for lentiviral RNAs was confirmed, whereas its affinity for HTLV-I RNAs was 1000-fold lower than for picornaviral RNAs. The BIAcore technology also revealed a significant affinity of the La autoantigen, previously described for its involvement in translational control of viral mRNAs, for HIV-1 and HTLV-I RNAs. Addition of recombinant PTB to in vitro translation experiments weakly enhanced translation initiation in the presence of HTLV-I IRES, suggesting that such an IRES requires additional trans-acting factors.

Calicheamicin−DNA Recognition: An Analysis of Seven Different Drug−DNA Complexes

A study of calicheamicin γ1I complexed to seven different recognition sites is presented. The recognition sites encompass a range of oligopyrimidine sites that present different topological features in the minor groove. Intermolecular NOE networks for the different calicheamicin-DNA complexes show that the drug binds in the same mode to each recognition site. Calicheamicin binding also induces a set of characteristic conformational changes in the DNA in each complex that maximize the complementarity of the fit between calicheamicin and the DNA. Based on an analysis of the different complexes as well as biochemical information on cleavage preferences, we propose that calicheamicin displays a shape-selective preference for pyrimidine tracts through an induced-fit mechanism. We predict that any carbohydrate that maintains the overall shape of the calicheamicin oligosaccharide will exhibit similar sequence selectivity. This hypothesis is supported by experiments on calicheamicin oligosaccharide analogues repo...

Nucleotide Excision Repair of the 5 S Ribosomal RNA Gene Assembled into a Nucleosome

A-175-base pair fragment containing the Xenopus borealis somatic 5 S ribosomal RNA gene was used as a model system to determine the effect of nucleosome assembly on nucleotide excision repair (NER) of the major UV photoproduct (cyclobutane pyrimidine dimer (CPD)) in DNA. Xenopus oocyte nuclear extracts were used to carry out repair in vitro on reconstituted, positioned 5 S rDNA nucleosomes. Nucleosome structure strongly inhibits NER at many CPD sites in the 5 S rDNA fragment while having little effect at a few sites. The time course of CPD removal at 35 different sites indicates that >85% of the CPDs in the naked DNA fragment have t(12) values <2 h, whereas <26% of the t(12) values in nucleosomes are <2 h, and 15% are >8 h. Moreover, removal of histone tails from these mononucleosomes has little effect on the repair rates. Finally, nucleosome inhibition of repair shows no correlation with the rotational setting of a 14-nucleotide-long pyrimidine tract located 30 base pairs from the nucleosome dyad. These results suggest that inhibition of NER by mononucleosomes is not significantly influenced by the rotational orientation of CPDs on the histone surface, and histone tails play little (or no) role in this inhibition.

Targeting of single-stranded DNA and RNA containing adjacent pyrimidine and purine tracts by triple helix formation with circular and clamp oligonucleotides.

The aim of this work was to construct an anti-messenger targeted to the pim-1 oncogene transcript, based on circular or clamp oligodeoxyribonucleotides. The formation of bimolecular triplexes by clamp or circular oligonucleotides was investigated using single-stranded targets of both DNA (5'-CCCTCCTTTGAAGAA-3') and RNA type (5'-CCCUCCUUUGAAGAA-3'). The third, 'Hoogsteen' strand of the triplex was represented by G,T-rich sequences. The secondary structures of the complexes were determined by thermal denaturation, circular dichroism and gel mobility shift experiments and shown to depend on the nature of the target strand. With DNA as target, the sequence of a clamp (or circular) oligonucleotide that formed the triple helix was 3'-GGGAGGAAACTTCTTTT-TTGTTGTTT-TT-GGTGGG-5', where the first TT dinucleotide (in italics) is a linker and the second TT (bold) represents the bridge through which the 'Hoogsteen' strand switches from one strand of the Watson-Crick duplex to the other, once the duplex is formed by the corresponding portion of the anti-messenger (underlined). The portion of the 'Hoogsteen' sequence of the triplex between the two TT dinucleotides binds to the 3' extremity of the target strand and runs parallel to it. The portion situated at the 5' end of the oligonucleotide switches to the purine tract of the complementary strand of the duplex and is antiparallel to it. In contrast, with RNA as target, for a branched clamp oligonucleotide that formed a triple helix over its entire length (5'-TTCTTCAAAGGAGGG-3' 3'-GGGTGGTTT-T-GTTGTT-5') the portion of the 'Hoogsteen' sequence that bound to the 3' extremity of the target strand had to be antiparallel to it.

PSF/p54(nrb) stimulates "jumping" of DNA topoisomerase I between separate DNA helices.

We have previously shown [Straub et al. (1998) J. Biol. Chem. 273, 26261] that the pyrimidine tract binding protein associated splicing factor PSF/p54(nrb) binds and stimulates DNA topoisomerase I. Here we show that cleavage and religation half-reactions of topoisomerase I are unaffected by PSF/p54(nrb), whereas the propensity of the enzyme to jump between separate DNA helices is stimulated. To demonstrate such an effect, topoisomerase I was first captured in suicidal cleavage of an oligonucleotide substrate. Subsequently, a cleavage/ligation equilibrium was established by adding a ligation donor under conditions allowing recleavage of the ligated substrate. Finally, a second oligonucleotide was added to the mixture, which also allowed suicidal cleavage by topoisomerase I, but did not accommodate the ligation donor of the first oligonucleotide. Thus, topoisomerase I was given the choice to engage in repeated cleavage/ligation cycles of the first oligonucleotide or to jump to the second suicide substrate and get trapped. PSF/p54(nrb) enhanced the cleavage rate of the second oligonucleotide (11-fold), suggesting that it stimulates the dissociation of topoisomerase I after ligation. Thus, stimulation of topoisomerase I catalysis by PSF/p54(nrb) seems to be affected by mobilization of the enzyme.

Isolation and characterization of medaka ribosomal protein S3a ( fte-1) cDNA and gene

Many ribosomal protein genes were cloned from different organisms. We describe here, for the first time, the isolation of the ribosomal protein S3a cDNA and gene from a teleost — the medaka ( Oryzias latipes). The cDNA sequence is 863 bp long and encodes an open reading frame of 266 amino acids. The gene is 2927 bp long and contains six introns and five introns. The levels of the S3a mRNA are elevated during embryonal development. Transcription of the gene was also detected in different tissues of adult medaka. At the 5′ untranslated region of the cDNA, the terminal pyrimidine tract, a common feature with all ribosomal protein genes, was found. snoRNA sequences were found in introns 3 and 5, similar to human and mouse U73b and U73a.

A novel type of splicing enhancer regulating adenovirus pre-mRNA splicing.

Splicing of the adenovirus IIIa pre-mRNA is subjected to a temporal regulation, such that efficient IIIa 3' splice site usage is confined to the late phase of the infectious cycle. Here we show that IIIa pre-mRNA splicing is activated more than 200-fold in nuclear extracts prepared from late adenovirus-infected cells (Ad-NE) compared to uninfected HeLa cell nuclear extracts (HeLa-NE). In contrast, splicing of the beta-globin pre-mRNA is repressed in Ad-NE. We constructed hybrid pre-mRNAs between IIIa and beta-globin in order to identify the minimal IIIa sequence element conferring enhanced splicing in Ad-NE. Using this approach, we show that the IIIa branch site/pyrimidine tract functions as a Janus element: it blocks splicing in HeLa-NE and functions as a splicing enhancer in Ad-NE. Therefore, we named this sequence the IIIa virus infection-dependent splicing enhancer (3VDE). This element is essential for regulated IIIa pre-mRNA splicing in Ad-NE and sufficient to confer an enhanced splicing phenotype to the beta-globin pre-mRNA in Ad-NE. We further show that the increase in IIIa splicing observed in Ad-NE is not accompanied by a similar increase in U2AF binding to the IIIa pyrimidine tract. This finding suggests that splicing activation by the 3VDE may operate without efficient U2AF interaction with the pre-mRNA. Importantly, this report represents the first description of a splicing enhancer that has evolved to function selectively in the context of a virus infection, a finding that adds a new level at which viruses may subvert the host cell RNA biosynthetic machinery to facilitate their own replication.

Isolation and molecular characterisation of the gene encoding the cytoplasmic ribosomal protein S28 in Prunus persica [L.] Batsch

RT-PCR was performed on peach (Prunus persica [L.] Batsch) RNA to isolate cDNAs corresponding to transcripts which are differentially expressed in leaves borne on basal and apical shoots. A gene was identified which was more highly expressed in the leaves of basal shoots, and codes for the cytoplasmic protein S28 present in the small ribosomal subunit. The 5' leader regions of RPS28 mRNAs were found to harbour 8-11 pyrimidine tracts, which suggested similarities to regulatory stretches that control the translation of mRNAs for ribosomal proteins in animals. The peach S28 is encoded by two intron-containing genes, which are both transcribed in mitotically active tissues such as developing leaves and roots. In situ hybridisation to shoot vegetative apices and the measurement of nucleus/nucleolus ratios indicated that RPS28 expression was confined to areas undergoing active cell division. The mature RPS28 mRNA was detected as a single species in actively dividing tissues such as apical tips, developing leaves, vegetative buds, stamens, developing fruits and roots. In contrast, accumulation of a precursor RNA, in the presence of the mature product, was found in fully expanded leaves and subtending stems, while only the precursor species was detected in several late-stage tissues. This phenomenon suggested that expression of the mature RNA is controlled at the level of splicing and turnover of the precursor RNA. This is similar to the mode of regulation of ribosomal protein genes in animals.

The carboxyl terminus of vertebrate poly(A) polymerase interacts with U2AF 65 to couple 3′-end processing and splicing

Although it has been established that the processing factors involved in pre-mRNA splicing and 3'-end formation can influence each other positively, the molecular basis of this coupling interaction was not known. Stimulation of pre-mRNA splicing by an adjacent cis-linked cleavage and polyadenylation site in HeLa cell nuclear extract is shown to occur at an early step in splicing, the binding of U2AF 65 to the pyrimidine tract of the intron 3' splice site. The carboxyl terminus of poly(A) polymerase (PAP) previously has been implicated indirectly in the coupling process. We demonstrate that a fusion protein containing the 20 carboxy-terminal amino acids of PAP, when tethered downstream of an intron, increases splicing efficiency and, like the entire 3'-end formation machinery, stimulates U2AF 65 binding to the intron. The carboxy-terminal domain of PAP makes a direct and specific interaction with residues 17-47 of U2AF 65, implicating this interaction in the coupling of splicing and 3'-end formation.