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Results for “RNA 3' Polyadenylation Signals”

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At least 19 recordsLinked to original sources

PubMed2004

Molecular cloning and characterization of a cDNA encoding the Paracoccidioides brasiliensis 135 ribosomal protein.

A 630 bp cDNA encoding an L35 ribosomal protein of Paracoccidioides brasiliensis, designated as Pbl35, was cloned from a yeast expression library. Pbl35 encodes a polypeptide of 125 amino acids, with a predicted molecular mass of 14.5 kDa and a pI of 11.0. The deduced PbL35 shows significant conservation in respect to other described ribosomal L35 proteins from eukaryotes and prokaryotes. Motifs of ribosomal proteins are present in PbL35, including a bipartite nuclear localization signal (NLS) that could be related to the protein addressing to the nucleolus for the ribosomal assembly. The mRNA for PbL35, about 700 nucleotides in length, is expressed at a high level in P. brasiliensis. The PbL35 and the deduced amino acid sequence constitute the first description of a ribosomal protein in P. brasiliensis. The cDNA was deposited in GenBank under accession number AF416509.

Amino Acid Sequence↗

PubMed2005

A 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hFip1, CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein.

We have investigated the role of the human papillomavirus type 16 (HPV-16) early untranslated region (3' UTR) in HPV-16 gene expression. We found that deletion of the early 3' UTR reduced the utilization of the early polyadenylation signal and, as a consequence, resulted in read-through into the late region and production of late L1 and L2 mRNAs. Deletion of the U-rich 3' half of the early 3' UTR had a similar effect, demonstrating that the 57-nucleotide U-rich region acted as an enhancing upstream element on the early polyadenylation signal. In accordance with this, the newly identified hFip1 protein, which has been shown to enhance polyadenylation through U-rich upstream elements, interacted specifically with the HPV-16 upstream element. This upstream element also interacted specifically with CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 early polyadenylation signal. Mutational inactivation of the early polyadenylation signal also resulted in increased late mRNA production. However, the effect was reduced by the activation of upstream cryptic polyadenylation signals, demonstrating the presence of additional strong RNA elements downstream of the early polyadenylation signal that direct cleavage and polyadenylation to this region of the HPV-16 genome. In addition, we identified a 3' splice site at genomic position 742 in the early region with the potential to produce E1 and E4 mRNAs on which the E1 and E4 open reading frames are preceded only by the suboptimal E6 AUG. These mRNAs would therefore be more efficiently translated into E1 and E4 than previously described HPV-16 E1 and E4 mRNAs on which E1 and E4 are preceded by both E6 and E7 AUGs.

3' Untranslated Regions↗

PubMed2003

An uncapped RNA suggests a model for Caenorhabditis elegans polycistronic pre-mRNA processing.

Polycistronic pre-mRNAs from Caenohabditis elegans operons are processed by internal cleavage and polyadenylation to create 3' ends of mature mRNAs. This is accompanied by trans-splicing with SL2 approximately 100 nucleotides downstream of the 3' end formation sites to create the 5' ends of downstream mRNAs. SL2 trans-splicing depends on a U-rich element (Ur), located approximately 70 nucleotides upstream of the trans-splice site in the intercistronic region (ICR), as well as a functional 3' end formation signal. Here we report the existence of a novel gene-length RNA, the Ur-RNA, starting just upstream of the Ur element. The expression of Ur-RNA is dependent on 3' end formation as well as on the presence of the Ur element, but does not require a trans-splice site. The Ur-RNA is not capped, and alteration of the location of the Ur element in either the 5' or 3' direction alters the location of the 5' end of the Ur-RNA. We propose that a 5' to 3' exonuclease degrades the precursor RNA following cleavage at the poly(A) site, stopping when it reaches the Ur element, presumably attributable to a bound protein. Part of the function of this protein can be performed by the MS2 coat protein. Recruitment of coat protein to the ICR in the absence of the Ur element results in accumulation of an RNA equivalent to Ur-RNA, and restores trans-splicing. Only SL1, however, is used. Therefore, coat protein is sufficient for blocking the exonuclease and thereby allowing formation of a substrate for trans-splicing, but it lacks the ability to recruit the SL2 snRNP. Our results also demonstrate that MS2 coat protein can be used as an in vivo block to an exonuclease, which should have utility in mRNA stability studies.

Animals↗

PubMed2005

The mouse Krüppel-like Factor 4 (Klf4) gene: four functional polyadenylation sites which are used in a cell-specific manner as revealed by testicular transcript analysis and multiple processed pseudogenes.

The transcription factor Krüppel-like factor 4 (Klf4) is involved in cell cycle arrest and terminal differentiation of many epithelial cell types. We have recently shown that Northern blot analysis of RNA from adult mouse testis revealed multiple Klf4 transcripts. In order to characterize these transcripts, we tested for alternative splicing events and looked for alternative transcriptional initiation and usage of different polyadenylation signals. We neither obtained evidence for alternative splicing nor found transcripts with novel 5' ends. However, we found striking differences in the 3' ends by RACE-PCR. These differences were, interestingly, due to the usage of four alternatively used polyadenylation signals (PAS). This high number of PAS is found in less than 1% of all genes. We show that testicular Sertoli cells exclusively use the first PAS, which is, notably, not canonical, while haploid germ cells rather use the more 3' located PAS-II-IV. The longer transcripts present in germ cells exhibit highly conserved putative binding motifs for proteins known to be important for translational regulation in germ cells. Moreover, we experimentally confirm an intron which was not described in a previous report on the Klf4 gene structure. Finally, we document six Klf4 pseudogenes most likely formed by L1-mediated retrotransposition, indicating germ line expression of Klf4. In summary, we show that mouse testicular cells make intensive use of alternative polyadenylation of Klf4 mRNA strongly suggesting translational regulation of the Klf4 message in spermatids.

3' Untranslated Regions↗

PubMed2003

Genomic organization and transcription of the human retinol dehydrogenase 10 (RDH10) gene.

A cDNA clone up-regulated in hydraulic lung edema in rabbit showed high similarity with human RDH10 mRNA, which encodes a protein involved in retinoic acid metabolism. We defined the organization of the human gene, which includes a unique transcriptional start site, a coding region with six translated exons and a 3' untranslated region containing at least two used polyadenylation sites. The two poly(A) signals are responsible for the production of the 3 and 4 kb RDH10 mRNA isoforms detected in several human tissues and cell lines.

3' Untranslated Regions↗

PubMed2006

[Polyadenylation signal-deficient retroviruses transformation of human gastric epithelial GES-1 cells].

OBJECTIVE: To verify if mutated polyadenylation signal retroviruses can produce viral-host readthrough transcripts (Rth) and have the ability to transform human gastric epithelial GES-1 cells, and to discuss the new functions of retroviruses in gastric cancer related gene research. METHODS: The polyadenylation signal-deficient retrovirus vector mutated by PCR site-directed mutagenesis was used to make polyadenylation signal-deficient retroviruses by PA317 packaging cells. The GES-1 cells were infected by the viruses and selected by G418. Viral-host readthrough RNAs were checked by Northern blot. The cell growth and soft agar assay were run to test the transformed cells. RESULTS: polyadenylation signal-deficient retroviruses could be packaged by PA317 packaging cells. The viruses had the ability to infect GES-1 cells. Northern blot analysis of viral RNA from infected pools and individual G418-resistant clones demonstrated that mutation of consensus LTR polyadenylation signals generated Rth viral RNA in the infected GES-1 cells. Phenotypic analysis results showed that the GES-1 cells infected with plyadenylation signal mutant viruses tended to grow in a cluster manner. Pools of PA317 cells infected with mutant viruses were able to form colonies in soft agar with a higher efficiency than control or uninfected cells. CONCLUSION: Host readthrough transcripts generated by polyadenylation signal mutant viruses may contribute to transformation GES-1 cell phenotypes. The mutant vectors and the method described in the present work may be useful as tools to trap and identify genes involved in retroviral insertion mediated cell transformation.

Animals↗

PubMed2006

An exploration of 3'-end processing signals and their tissue distribution in Oryza sativa.

The 3' untranslated regions deeply affect many properties of eukaryotic mRNA. In plants, the polyadenine control signals contained in these regions seem to be more variable than of mammals. Three cDNA libraries derived from the leaf, endosperm and stem tissues of rice were sequenced from the 3'-end. Of the 9911 transcripts analyzed, 5723 unique transcripts were identified from the leaf sequences, 2934 from the endosperm and 1254 from the stem. The information entropy and two statistical methods were used to compile a list of rice poly(A) control signals. Based on their distribution, these signals can be roughly grouped into far-upstream element (FUE), near-upstream element (NUE), T-rich region (TRE) and downstream element (DE). The distribution of rice conserved regions is similar to the previous model from Arabidopsis and yeast, with a few differences in word constructions. Interestingly, we also found the word distributions were diverse in the cleavage site of downstream sequences of different rice tissues. The signal bias in downstream sequences may lead mRNA to be differently cleaved in different rice tissues.

3' Untranslated Regions↗

PubMed2006

Expression of TPK1 and TPK2 genes encoding PKA catalytic subunits during growth and morphogenesis in Candida albicans.

The transcript levels of Candida albicans TPK1 and TPK2 genes, encoding PKA catalytic subunits, as well as phosphotransferase activity, were measured in the parental strain CAI4 and in homozygous tpk1Delta and tpk2Delta mutants during vegetative growth and during yeast-to-mycelial transition in N-acetylglucosamine liquid inducing medium at 37 degrees C. We observed two TPK2 transcripts, a major one of 1.8 kb and a minor one of 1.4 kb, and established by 3'-RACE that they originate from the recognition of the three polyadenylation signals present in the 3' untranslated region of the gene. During vegetative growth of CAI4 strain, the expression profiles of TPK1 and TPK2 varied similarly, reaching maximal expression at the late logarithmic phase. TPK1 mRNA levels were lower than those of TPK2 at all stages measured. In the corresponding homozygous tpk mutants, mRNA levels and the expression patterns of TPK1 and TPK2 were similar to those of CAI4, suggesting that the loss of one catalytic isoform is not compensated by overexpression of the other. Changes in PKA specific activity roughly correlated with fluctuations of mRNA expression levels. During yeast-to-mycelial transition, a sharp increase in TPK1 mRNA levels and in PKA-specific activity correlated with the onset of germ-tube formation in strain tpk2Delta. We also showed that tpk1Delta strain exhibited a delayed morphogenetic shift in comparison with CAI4 and tpk2Delta strains in several liquid inducing media, reinforcing the idea that Tpk1p is important for faster germ-tube appearance.

3' Untranslated Regions↗

PubMed2006

Transcriptional origin of Kaposi's sarcoma-associated herpesvirus microRNAs.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 11 distinct microRNAs, all of which are found clustered within the major latency-associated region of the KSHV genome in the same transcriptional orientation. Because the KSHV microRNAs are all expressed in latently infected cells and are largely unaffected by induction of lytic replication, it appeared probable that they would be processed out of KSHV transcripts that are derived from a latent promoter(s) present in this region. Here, we define three latent transcripts, derived from two distinct KSHV latent promoters, that function as both KSHV primary microRNA precursors and as kaposin pre-mRNAs. These activities require the readthrough of a leaky viral polyadenylation signal located at nucleotide 122070 in the KSHV genome. In contrast, recognition of this polyadenylation signal gives rise to previously identified mRNAs that encode the KSHV open reading frames (ORFs) 71, 72 and 73 proteins as well as a novel unspliced KSHV mRNA that encodes only ORF72 and ORF71. Thus, transcripts initiating at the two latent promoters present in the KSHV latency-associated region can undergo two entirely distinct fates, i.e., processing to give a kaposin mRNA and viral microRNAs on the one hand or expression as KSHV ORF71, ORF72, or ORF73 mRNAs on the other, depending on whether the viral polyadenylation site located at position 122070 is ignored or recognized, respectively.

Cell Line↗

PubMed2002

Reexamining the polyadenylation signal: were we wrong about AAUAAA?

Polyadenylation is the process by which most eukaryotic mRNAs form their 3' ends. It was long held that polyadenylation required the sequence AAUAAA and that 90% of mRNAs had AAUAAA within 30 nucleotides of the site of poly(A) addition. More recent studies, aided by computer analysis of sequences made available in GenBank and expressed sequence tag (EST) databases, have suggested that the actual incidence of AAUAAA is much lower, perhaps as low as 50-60%. Reproductive biologists have long recognized that a large number of mRNAs in male germ cells of mammals lack AAUAAA but are otherwise normally polyadenylated. Recent research in our laboratory has uncovered a new form of an essential polyadenylation protein, tauCstF-64, that is most highly expressed in male germ cells, and to a smaller extent in the brain, and which we propose plays a significant role in AAUAAA-independent mRNA polyadenylation in germ cells.

Animals↗

PubMed2004

Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase.

In mammals, polyadenylation of mRNA precursors (pre-mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF). CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal. Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF. hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA. Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner. hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA. These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity.

Amino Acid Motifs↗

PubMed2006

Systematic variation in mRNA 3'-processing signals during mouse spermatogenesis.

Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3'-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3'-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3'-processing characteristics in the testicular samples, compared to control sets of widely used 3'-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3'-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3'-untranslated regions (3'-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3'-UTR truncation and no significant difference in 3'-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3'-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis.

3' Untranslated Regions↗

PubMed2005

RNA turnover: unexpected consequences of being tailed.

In eukaryotic cells, the 3' poly(A) tails found on mRNA influence their stability and translation. The discovery of a second nuclear poly(A) polymerase complex has fueled a series of reports defining a new and unexpected role for 3' end poly(A) tails in the nuclear surveillance and turnover of noncoding RNAs and intergenic mRNAs of unknown function.

Eukaryotic Cells↗

PubMed2005

Alternative splicing of human insulin-degrading enzyme yields a novel isoform with a decreased ability to degrade insulin and amyloid beta-protein.

Deletion of insulin-degrading enzyme (IDE) in mice causes accumulation of cerebral amyloid beta-protein (Abeta), hyperinsulinemia, and glucose intolerance. Together with genetic linkage and allelic association of IDE to Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), these findings suggest that IDE hypofunction could mediate human disease. To date, no coding mutations have been found in the canonical isoform of IDE, suggesting that pathological mutations could exist in undiscovered exons or regulatory regions, including untranslated regions (UTRs). However, neither isoforms arising from alternative splicing nor the UTRs have been described. Here, we systematically characterize human IDE mRNAs, identify a novel splice form, and compare its subcellular distribution, kinetic properties, and ability to degrade Abeta to the known isoform. Six distinct human IDE transcripts were identified, with most of the variance attributable to alternative polyadenylation sites. In the novel spliceoform, an exon we designate "15b" replaces the canonical exon "15a", and the resultant variant is widely expressed. Subcellular fractionation, immunofluorescent confocal microscopy, and immunogold-electron microscopy reveal that the 15b-IDE protein occurs in both cytosol and mitochondria. Organelle targeting of both isoforms is determined by which of two translation start sites is used, and only those isoforms utilizing the second site regulate levels of secreted Abeta. 15b-IDE can exist as a heterodimer with the 15a isoform or as a homodimer. The apparent K(m) values of recombinant 15b-IDE for both insulin and Abeta are significantly higher and the k(cat) and catalytic efficiency markedly lower than those of 15a-IDE. In accord, cells coexpressing beta-amyloid precursor protein (APP) and 15b-IDE accumulated significantly more Abeta in their media than those expressing APP and 15a-IDE. Our results identify a novel, catalytically inefficient form of IDE expressed in brain and non-neural tissues and recommend novel regions of the IDE gene in which to search for mutations predisposing patients to AD and DM2.

Alternative Splicing↗

PubMed2006

Oligomerization of EDEN-BP is required for specific mRNA deadenylation and binding.

BACKGROUND INFORMATION: mRNA deadenylation [shortening of the poly(A) tail] is often triggered by specific sequence elements present within mRNA 3' untranslated regions and generally causes rapid degradation of the mRNA. In vertebrates, many of these deadenylation elements are called AREs (AU-rich elements). The EDEN (embryo deadenylation element) sequence is a Xenopus class III ARE. EDEN acts by binding a specific factor, EDEN-BP (EDEN-binding protein), which in turn stimulates deadenylation. RESULTS: We show here that EDEN-BP is able to oligomerize. A 27-amino-acid region of EDEN-BP was identified as a key domain for oligomerization. A mutant of EDEN-BP lacking this region was unable to oligomerize, and a peptide corresponding to this region competitively inhibited the oligomerization of full-length EDEN-BP. Impairing oligomerization by either of these two methods specifically abolished EDEN-dependent deadenylation. Furthermore, impairing oligomerization inhibited the binding of EDEN-BP to its target RNA, demonstrating a strong coupling between EDEN-BP oligomerization and RNA binding. CONCLUSIONS: These data, showing that the oligomerization of EDEN-BP is required for binding of the protein on its target RNA and for EDEN-dependent deadenylation in Xenopus embryos, will be important for the identification of cofactors required for the deadenylation process.

Adenosine Monophosphate↗

PubMed2004

Molecular cloning and characterization of a cDNA encoding the N-acetyl-beta-D-glucosaminidase homologue of Paracoccidioides brasiliensis.

A cDNA encoding the N-acetyl-beta-D-glucosaminidase (NAG) protein of Paracoccidioides brasiliensis, Pb NAG1, was cloned and characterized. The 2663-nucleotide sequence of the cDNA consisted of a single open reading frame encoding a protein with a predicted molecular mass of 64.73 kDa and an isoeletric point of 6.35. The predicted protein includes a putative 30-amino-acid signal peptide. The protein as a whole shares considerable sequence similarity with 'classic' NAG. The primary sequence of Pb NAG1 was used to infer phylogenetic relationships. The amino acid sequence of Pb NAG1 has 45, 31 and 30% identity, respectively, with homologous sequences from Trichoderma harzianum, Aspergillus nidulans and Candida albicans. In particular, striking homology was observed with the active site regions of the glycosyl hydrolase group of proteins (family 20). The expected active site consensus motif G X D E and catalytic Asp and Glu residues at positions 373 and 374 were found, reinforcing that Pb NAG1 belongs to glycosyl hydrolase family 20. The nucleotide sequence of Pb nag1 and its flanking regions have been deposited, along with the amino acid sequence of the deduced protein, in GenBank under accession number AF419158.

5' Untranslated Regions↗

PubMed2004

A probabilistic model of 3' end formation in Caenorhabditis elegans.

The 3' ends of mRNAs terminate with a poly(A) tail. This post-transcriptional modification is directed by sequence features present in the 3'-untranslated region (3'-UTR). We have undertaken a computational analysis of 3' end formation in Caenorhabditis elegans. By aligning cDNAs that diverge from genomic sequence at the poly(A) tract, we accurately identified a large set of true cleavage sites. When there are many transcripts aligned to a particular locus, local variation of the cleavage site over a span of a few bases is frequently observed. We find that in addition to the well-known AAUAAA motif there are several regions with distinct nucleotide compositional biases. We propose a generalized hidden Markov model that describes sequence features in C.elegans 3'-UTRs. We find that a computer program employing this model accurately predicts experimentally observed 3' ends even when there are multiple AAUAAA motifs and multiple cleavage sites. We have made available a complete set of polyadenylation site predictions for the C.elegans genome, including a subset of 6570 supported by aligned transcripts.

3' Untranslated Regions↗

PubMed2006

GRSDB: a database of quadruplex forming G-rich sequences in alternatively processed mammalian pre-mRNA sequences.

Guanine-rich nucleic acids are known to form highly stable G-quadruplex structures, also known as G-quartets. Recently, there has been a tremendous amount of interest in studying G-quadruplexes owing to the realization of their biological importance. G-rich sequences (GRSs) capable of forming G-quadruplexes are found in the vicinity of polyadenylation regions and are involved in regulating 3' end processing of mammalian pre-mRNAs. G-rich motifs are also known to play an important role in alternative, tissue-specific splicing by interacting with hnRNP H protein subfamily. Whether quadruplex structure directly plays a role in regulating RNA processing events requires further investigation. To date there has not been a comprehensive effort to study G-quadruplexes near RNA processing sites. We have applied a computational approach to map putative Quadruplex forming GRSs within the transcribed regions of a large number of alternatively processed human and mouse gene sequences that were obtained as fully annotated entries from GenBank and RefSeq. We have used the computed data to build the GRSDB database that provides a unique avenue for studying G-quadruplexes in the context of RNA processing sites. GRSDB website offers visual comparison of G-quadruplex distribution patterns among all the alternative RNA products of a gene with the help of dynamic graphics. At present, GRSDB contains data from 1310 human and mouse genes, of which 1188 are alternatively processed. It has a total of 379,223 predicted G-quadruplexes, of which 54,252 are near RNA processing sites. GRSDB is a good resource for researchers interested in investigating the functional relevance of G-quadruplexes, especially in the context of alternative RNA processing. It can be accessed at http://bioinformatics.ramapo.edu/grsdb/.

Alternative Splicing↗