Showing papers on "Intron published in 2007"

RNAmmer: consistent and rapid annotation of ribosomal RNA genes

Abstract: The publication of a complete genome sequence is usually accompanied by annotations of its genes. In contrast to protein coding genes, genes for ribosomal RNA (rRNA) are often poorly or inconsistently annotated. This makes comparative studies based on rRNA genes difficult. We have therefore created computational predictors for the major rRNA species from all kingdoms of life and compiled them into a program called RNAmmer. The program uses hidden Markov models trained on data from the 5S ribosomal RNA database and the European ribosomal RNA database project. A pre-screening step makes the method fast with little loss of sensitivity, enabling the analysis of a complete bacterial genome in less than a minute. Results from running RNAmmer on a large set of genomes indicate that the location of rRNAs can be predicted with a very high level of accuracy. Novel, unannotated rRNAs are also predicted in many genomes. The software as well as the genome analysis results are available at the CBS web server.

A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains

Abstract: Noncoding RNA species play a diverse set of roles in the eukaryotic cell. While much recent attention has focused on smaller RNA species, larger noncoding transcripts are also thought to be highly abundant in mammalian cells. To search for large noncoding RNAs that might control gene expression or mRNA metabolism, we used Affymetrix expression arrays to identify polyadenylated RNA transcripts displaying nuclear enrichment. This screen identified no more than three transcripts; XIST, and two unique noncoding nuclear enriched abundant transcripts (NEAT) RNAs strikingly located less than 70 kb apart on human chromosome 11: NEAT1, a noncoding RNA from the locus encoding for TncRNA, and NEAT2 (also known as MALAT-1). While the two NEAT transcripts share no significant homology with each other, each is conserved within the mammalian lineage, suggesting significant function for these noncoding RNAs. NEAT2 is extraordinarily well conserved for a noncoding RNA, more so than even XIST. Bioinformatic analyses of publicly available mouse transcriptome data support our findings from human cells as they confirm that the murine homologs of these noncoding RNAs are also nuclear enriched. RNA FISH analyses suggest that these noncoding RNAs function in mRNA metabolism as they demonstrate an intimate association of these RNA species with SC35 nuclear speckles in both human and mouse cells. These studies show that one of these transcripts, NEAT1 localizes to the periphery of such domains, whereas the neighboring transcript, NEAT2, is part of the long-sought polyadenylated component of nuclear speckles. Our genome-wide screens in two mammalian species reveal no more than three abundant large non-coding polyadenylated RNAs in the nucleus; the canonical large noncoding RNA XIST and NEAT1 and NEAT2. The function of these noncoding RNAs in mRNA metabolism is suggested by their high levels of conservation and their intimate association with SC35 splicing domains in multiple mammalian species.

Processing of intronic microRNAs

Abstract: The majority of human microRNA (miRNA) loci are located within intronic regions and are transcribed by RNA polymerase II as part of their hosting transcription units. The primary transcripts are cleaved by Drosha to release ∼70 nt pre-miRNAs that are subsequently processed by Dicer to generate mature ∼22 nt miRNAs. It is generally believed that intronic miRNAs are released by Drosha from excised introns after the splicing reaction has occurred. However, our database searches and experiments indicate that intronic miRNAs can be processed from unspliced intronic regions before splicing catalysis. Intriguingly, cleavage of an intron by Drosha does not significantly affect the production of mature mRNA, suggesting that a continuous intron may not be required for splicing and that the exons may be tethered to each other. Hence, Drosha may cleave intronic miRNAs between the splicing commitment step and the excision step, thereby ensuring both miRNA biogenesis and protein synthesis from a single primary transcript. Our study provides a novel example of eukaryotic gene organization and RNA-processing control.

Specialization and evolution of endogenous small RNA pathways.

Abstract: The specificity of RNA silencing is conferred by small RNA guides that are processed from structured RNA or dsRNA. The core components for small RNA biogenesis and effector functions have proliferated and specialized in eukaryotic lineages, resulting in diversified pathways that control expression of endogenous and exogenous genes, invasive elements and viruses, and repeated sequences. Deployment of small RNA pathways for spatiotemporal regulation of the transcriptome has shaped the evolution of eukaryotic genomes and contributed to the complexity of multicellular organisms.

Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements

Abstract: The human and mouse genomes share a number of long, perfectly conserved nucleotide sequences, termed ultraconserved elements. Whereas these regions can act as transcriptional enhancers when upstream of genes, those within genes are less well understood. In particular, the function of ultraconserved elements that overlap alternatively spliced exons of genes encoding RNA-binding proteins is unknown. Here we report that in every member of the human SR family of splicing regulators, highly or ultraconserved elements are alternatively spliced, either as alternative 'poison cassette exons' containing early in-frame stop codons, or as alternative introns in the 3' untranslated region. These alternative splicing events target the resulting messenger RNAs for degradation by means of an RNA surveillance pathway called nonsense-mediated mRNA decay. Mouse orthologues of the human SR proteins exhibit the same unproductive splicing patterns. Three SR proteins have been previously shown to direct splicing of their own transcripts, and one of these is known to autoregulate its expression by coupling alternative splicing with decay; our results suggest that unproductive splicing is important for regulation of the entire SR family. We find that unproductive splicing associated with conserved regions has arisen independently in different SR genes, suggesting that splicing factors may readily acquire this form of regulation.

Genomic analysis of human microRNA transcripts

Abstract: MicroRNAs (miRNAs) are important genetic regulators of development, differentiation, growth, and metabolism. The mammalian genome encodes ≈500 known miRNA genes. Approximately 50% are expressed from non-protein-coding transcripts, whereas the rest are located mostly in the introns of coding genes. Intronic miRNAs are generally transcribed coincidentally with their host genes. However, the nature of the primary transcript of intergenic miRNAs is largely unknown. We have performed a large-scale analysis of transcription start sites, polyadenylation signals, CpG islands, EST data, transcription factor-binding sites, and expression ditag data surrounding intergenic miRNAs in the human genome to improve our understanding of the structure of their primary transcripts. We show that a significant fraction of primary transcripts of intergenic miRNAs are 3–4 kb in length, with clearly defined 5′ and 3′ boundaries. We provide strong evidence for the complete transcript structure of a small number of human miRNAs.

A Contemporary View of Coronavirus Transcription

Abstract: Coronaviruses are a family of enveloped, plus-stranded RNA viruses with helical nucleocapsids and extraordinarily large genomes. The hallmark of coronavirus transcription is the production of multiple subgenomic mRNAs that contain sequences corresponding to both ends of the genome. (Transcription is

Alternative Splicing of Pre-Messenger RNAs in Plants in the Genomic Era

Abstract: Primary transcripts (precursor-mRNAs) with introns can undergo alternative splicing to produce multiple transcripts from a single gene by differential use of splice sites, thereby increasing the transcriptome and proteome complexity within and between cells and tissues. Alternative splicing in plants is largely an unexplored area of gene expression, as this phenomenon used to be considered rare. However, recent genome-wide computational analyses have revealed that alternative splicing in flowering plants is far more prevalent than previously thought. Interestingly, pre-mRNAs of many spliceosomal proteins, especially serine/arginine-rich (SR) proteins, are extensively alternatively spliced. Furthermore, stresses have a dramatic effect on alternative splicing of pre-mRNAs including those that encode many spliceosomal proteins. Although the mechanisms that regulate alternative splicing in plants are largely unknown, several reports strongly suggest a key role for SR proteins in spliceosome assembly and regulated splicing. Recent studies suggest that alternative splicing in plants is an important posttranscriptional regulatory mechanism in modulating gene expression and eventually plant form and function.

Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay

Abstract: Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additional examples identified >50 such exons in genes with a variety of functions. These exons are unusually frequent in genes that encode splicing activators and are unexpectedly enriched in the so-called “ultraconserved” elements in the mammalian lineage. Further analysis show that NMD of mRNAs for splicing activators such as SR proteins is triggered by splicing activation events, whereas NMD of the mRNAs for negatively acting hnRNP proteins is triggered by splicing repression, a polarity consistent with widespread homeostatic control of splicing regulator gene expression. We suggest that the extreme genomic conservation surrounding these regulatory splicing events within splicing factor genes demonstrates the evolutionary importance of maintaining tightly tuned homeostasis of RNA-binding protein levels in the vertebrate cell.

Different levels of alternative splicing among eukaryotes

Abstract: Alternative splicing increases transcriptome and proteome diversification. Previous analyses aiming at comparing the rate of alternative splicing between different organisms provided contradicting results. These contradicting results were attributed to the fact that both analyses were dependent on the expressed sequence tag (EST) coverage, which varies greatly between the tested organisms. In this study we compare the level of alternative splicing among eight different organisms. By employing an EST independent approach we reveal that the percentage of genes and exons undergoing alternative splicing is higher in vertebrates compared with invertebrates. We also find that alternative exons of the skipping type are flanked by longer introns compared to constitutive ones, whereas alternative 5' and 3' splice sites events are generally not. In addition, although the regulation of alternative splicing and sizes of introns and exons have changed during metazoan evolution, intron retention remained the rarest type of alternative splicing, whereas exon skipping is more prevalent and exhibits a slight increase, from invertebrates to vertebrates. The difference in the level of alternative splicing suggests that alternative splicing may contribute greatly to the mammal higher level of phenotypic complexity, and that accumulation of introns confers an evolutionary advantage as it allows increasing the number of alternative splicing forms.

Control of alternative RNA splicing and gene expression by eukaryotic riboswitches.

Abstract: Bacteria make extensive use of riboswitches to sense metabolites and control gene expression, and typically do so by modulating premature transcription termination or translation initiation. The most widespread riboswitch class known in bacteria responds to the coenzyme thiamine pyrophosphate (TPP), which is a derivative of vitamin B1. Representatives of this class have also been identified in fungi and plants, where they are predicted to control messenger RNA splicing or processing. We examined three TPP riboswitches in the filamentous fungus Neurospora crassa, and found that one activates and two repress gene expression by controlling mRNA splicing. A detailed mechanism involving riboswitch-mediated base-pairing changes and alternative splicing control was elucidated for precursor NMT1 mRNAs, which code for a protein involved in TPP metabolism. These results demonstrate that eukaryotic cells employ metabolite-binding RNAs to regulate RNA splicing events that are important for the control of key biochemical processes.

RNA editing of the microRNA-151 precursor blocks cleavage by the Dicer–TRBP complex

Abstract: MicroRNAs (miRNAs) mediate translational repression or degradation of their target messenger RNAs by RNA interference (RNAi). The primary transcripts of miRNA genes (pri-miRNAs) are sequentially processed by the nuclear Drosha–DGCR8 complex to approximately 60–70 nucleotide (nt) intermediates (pre-miRNAs) and then by the cytoplasmic Dicer–TRBP complex to approximately 20–22 nt mature miRNAs. Certain pri-miRNAs are subject to RNA editing that converts adenosine to inosine (A → I RNA editing); however, the fate of edited pri-miRNAs is mostly unknown. Here, we provide evidence that RNA editing of pri-miR-151 results in complete blockage of its cleavage by Dicer and accumulation of edited pre-miR-151 RNAs. Our results indicate that A → I conversion at two specific positions of the pre-miRNA foldback structure can affect its interaction with the Dicer–TRBP complex, showing a new regulatory role of A → I RNA editing in miRNA biogenesis.

Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins: regulation by hormones and stresses

Abstract: Precursor mRNAs with introns can undergo alternative splicing (AS) to produce structurally and functionally different proteins from the same gene. Here, we show that the pre-mRNAs of Arabidopsis genes that encode serine/arginine-rich (SR) proteins, a conserved family of splicing regulators in eukaryotes, are extensively alternatively spliced. Remarkably about 95 transcripts are produced from only 15 genes, thereby increasing the complexity of the SR gene family transcriptome by six-fold. The AS of some SR genes is controlled in a developmental and tissue-specific manner. Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) dramatically altered the AS of pre-mRNAs of several SR genes, whereas hormones altered the splicing of only three SR genes. These results indicate that abiotic stresses regulate the AS of the pre-mRNAs of SR genes to produce different isoforms of SR proteins that are likely to have altered function(s) in pre-mRNA splicing. Sequence analysis of splice variants revealed that predicted proteins from a majority of these variants either lack one or more modular domains or contain truncated domains. Because of the modular nature of the various domains in SR proteins, the proteins produced from splice variants are likely to have distinct functions. Together our results indicate that Arabidopsis SR genes generate surprisingly large transcriptome complexity, which is altered by stresses and hormones.

Spliced leader RNA trans-splicing in dinoflagellates.

Abstract: Through the analysis of hundreds of full-length cDNAs from fifteen species representing all major orders of dinoflagellates, we demonstrate that nuclear-encoded mRNAs in all species, from ancestral to derived lineages, are trans-spliced with the addition of the 22-nt conserved spliced leader (SL), DCCGUAGCCAUUUUGGCUCAAG (D = U, A, or G), to the 5′ end. SL trans-splicing has been documented in a limited but diverse number of eukaryotes, in which this process makes it possible to translate polycistronically transcribed nuclear genes. In SL trans-splicing, SL-donor transcripts (SL RNAs) contain two functional domains: an exon that provides the SL for mRNA and an intron that contains a spliceosomal (Sm) binding site. In dinoflagellates, SL RNAs are unusually short at 50–60 nt, with a conserved Sm binding motif (AUUUUGG) located in the SL (exon) rather than the intron. The initiation nucleotide is predominantly U or A, an unusual feature that may affect capping, and hence the translation and stability of the recipient mRNA. The core SL element was found in mRNAs coding for a diverse array of proteins. Among the transcripts characterized were three homologs of Sm-complex subunits, indicating that the role of the Sm binding site is conserved, even if the location on the SL is not. Because association with an Sm-complex often signals nuclear import for U-rich small nuclear RNAs, it is unclear how this Sm binding site remains on mature mRNAs without impeding cytosolic localization or translation of the latter.

Riboswitch Control of Gene Expression in Plants by Splicing and Alternative 3′ End Processing of mRNAs

Abstract: The most widespread riboswitch class, found in organisms from all three domains of life, is responsive to the vitamin B(1) derivative thiamin pyrophosphate (TPP). We have established that a TPP-sensing riboswitch is present in the 3' untranslated region (UTR) of the thiamin biosynthetic gene THIC of all plant species examined. The THIC TPP riboswitch controls the formation of transcripts with alternative 3' UTR lengths, which affect mRNA accumulation and protein production. We demonstrate that riboswitch-mediated regulation of alternative 3' end processing is critical for TPP-dependent feedback control of THIC expression. Our data reveal a mechanism whereby metabolite-dependent alteration of RNA folding controls splicing and alternative 3' end processing of mRNAs. These findings highlight the importance of metabolite sensing by riboswitches in plants and further reveal the significance of alternative 3' end processing as a mechanism of gene control in eukaryotes.

Disease-associated intronic variants in the ErbB4 gene are related to altered ErbB4 splice-variant expression in the brain in schizophrenia

Abstract: The neuregulin 1 (NRG1) receptor, ErbB4, has been identified as a potential risk gene for schizophrenia. HER4/ErbB4 is a receptor tyrosine kinase whose transcript undergoes alternative splicing in the brain. Exon 16 encodes isoforms containing a metalloprotease cleavable extracellular domain (JM-a), exon 15 for a cleavage resistant form (JM-b) and exon 26 for a cytoplasmic domain (CYT-1) with a phosphotidylinositol-3 kinase (PI3K) binding site. Disease-associated variants in the ErbB4 gene are intronic and implicate altered splicing of the gene. We examined ErbB4 splice-variant gene expression in the hippocampus and dorsolateral prefrontal cortex (DLPFC) in schizophrenia using qPCR and investigated whether expression levels are associated with previously reported genomic risk variants in ErbB4 in a large cohort of human brains. In the DLPFC, we confirmed previous observations, in a separate cohort, that mRNA for ErbB4 splice isoforms containing exon 16 (JM-a) and exon 26 (CYT-1) are significantly elevated in patients with schizophrenia. A main effect of genotype was observed in the DLPFC and hippocampus at a single risk SNP located in intron 12 (rs4673628) on isoforms containing exon 16 (JM-a). We also found that three intronic risk SNPs (rs7598440, rs707284, rs839523) and a core-risk haplotype surrounding exon 3 are strongly associated with elevated expression of splice variants containing exon 26 (CYT-1). These findings suggest that dysregulation of splice-variant specific expression of ErbB4 in the brain underlies the genetic association of the gene with schizophrenia and that the NRG1/ErbB4 signaling pathway may be an important genetic network involved in the pathogenesis of the disease.

RNA chaperones, RNA annealers and RNA helicases.

Abstract: RNA molecules face difficulties when folding into their native structures. In the cell, proteins can assist RNAs in reaching their functionally active states by binding and stabilizing a specific structure or, in a quite opposite way, by interacting in a non-specific manner. These proteins can either facilitate RNA-RNA interactions in a reaction termed RNA annealing, or they can resolve non-functional inhibitory structures. The latter is defined as "RNA chaperone activity" and is the main topic of this review. Here we define RNA chaperone activity in a stringent way and we review those proteins for which RNA chaperone activity has been clearly demonstrated. These proteins belong to quite diverse families such as hnRNPs, histone-like proteins, ribosomal proteins, cold shock domain proteins and viral nucleocapsid proteins. DExD/H-box containing RNA helicases are discussed as a special family of enzymes that restructure RNA or RNPs in an ATP-dependent manner. We further address the different mechanisms RNA chaperones might use to promote folding including the recently proposed theory of protein disorder as a key element in triggering RNA-protein interactions. Finally, we present a new website for proteins with RNA chaperone activity which compiles all the information on these proteins with the perspective to promote the understanding of their activity.

Genome-Wide Analysis of mRNA Decay Rates and Their Determinants in Arabidopsis thaliana

Abstract: To gain a global view of mRNA decay in Arabidopsis thaliana, suspension cell cultures were treated with a transcriptional inhibitor, and microarrays were used to measure transcript abundance over time. The deduced mRNA half-lives varied widely, from minutes to >24 h. Three features of the transcript displayed a correlation with decay rates: (1) genes possessing at least one intron produce mRNA transcripts significantly more stable than those of intronless genes, and this was not related to overall length, sequence composition, or number of introns; (2) various sequence elements in the 3′ untranslated region are enriched among short- and long-lived transcripts, and their multiple occurrence suggests combinatorial control of transcript decay; and (3) transcripts that are microRNA targets generally have short half-lives. The decay rate of transcripts correlated with subcellular localization and function of the encoded proteins. Analysis of transcript decay rates for genes encoding orthologous proteins between Arabidopsis, yeast, and humans indicated that yeast and humans had a higher percentage of transcripts with shorter half-lives and that the relative stability of transcripts from genes encoding proteins involved in cell cycle, transcription, translation, and energy metabolism is conserved. Comparison of decay rates with changes in transcript abundance under a variety of abiotic stresses reveal that a set of transcription factors are downregulated with similar kinetics to decay rates, suggesting that inhibition of their transcription is an important early response to abiotic stress.

The mRNA-like noncoding RNA Gomafu constitutes a novel nuclear domain in a subset of neurons

Abstract: Recent transcriptome analyses have revealed that a large body of noncoding regions of mammalian genomes are actually transcribed into RNAs. Our understanding of the molecular features of these noncoding RNAs is far from complete. We have identified a novel mRNA-like noncoding gene, named Gomafu, which is expressed in a distinct set of neurons in the mouse nervous system. Interestingly, spliced mature Gomafu RNA is localized to the nucleus despite its mRNA-like characteristics, which usually act as potent export signals to the cytoplasm. Within the nucleus, Gomafu RNA is detected as numerous spots that do not colocalize with known nuclear domain markers. Gomafu RNA is extremely insoluble and remains intact after nuclear matrix preparation. Furthermore, heterokaryon assays revealed that Gomafu RNA does not shuttle between the nucleus and cytoplasm, but is retained in the nucleus after its transcription. We propose that Gomafu RNA represents a novel family of mRNA-like noncoding RNA that constitutes a cell-type-specific component of the nuclear matrix.

The Pentatricopeptide Repeat Gene OTP43 Is Required for trans-Splicing of the Mitochondrial nad1 Intron 1 in Arabidopsis thaliana

Abstract: The mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) is a large protein complex formed from both nuclearly and mitochondrially encoded subunits. Subunit ND1 is encoded by a mitochondrial gene comprising five exons, and the mature transcript requires four RNA splicing events, two of which involve trans-splicing independently transcribed RNAs. We have identified a nuclear gene (OTP43) absolutely required for trans-splicing of intron 1 (and only intron 1) of Arabidopsis thaliana nad1 transcripts. This gene encodes a previously uncharacterized pentatricopeptide repeat protein. Mutant Arabidopsis plants with a disrupted OTP43 gene do not present detectable mitochondrial Complex I activity and show severe defects in seed development, germination, and to a lesser extent in plant growth. The alternative respiratory pathway involving alternative oxidase is significantly induced in the mutant.

Comparative genomic analysis of prion genes

Abstract: The homologues of human disease genes are expected to contribute to better understanding of physiological and pathogenic processes. We made use of the present availability of vertebrate genomic sequences, and we have conducted the most comprehensive comparative genomic analysis of the prion protein gene PRNP and its homologues, shadow of prion protein gene SPRN and doppel gene PRND, and prion testis-specific gene PRNT so far. While the SPRN and PRNP homologues are present in all vertebrates, PRND is known in tetrapods, and PRNT is present in primates. PRNT could be viewed as a TE-associated gene. Using human as the base sequence for genomic sequence comparisons (VISTA), we annotated numerous potential cis-elements. The conserved regions in SPRN s harbour the potential Sp1 sites in promoters (mammals, birds), C-rich intron splicing enhancers and PTB intron splicing silencers in introns (mammals, birds), and hsa-miR-34a sites in 3'-UTR s (eutherians). We showed the conserved PRNP upstream regions, which may be potential enhancers or silencers (primates, dog). In the PRNP 3'-UTR s, there are conserved cytoplasmic polyadenylation element sites (mammals, birds). The PRND core promoters include highly conserved CCAAT, CArG and TATA boxes (mammals). We deduced 42 new protein primary structures, and performed the first phylogenetic analysis of all vertebrate prion genes. Using the protein alignment which included 122 sequences, we constructed the neighbour-joining tree which showed four major clusters, including shadoos, shadoo2s and prion protein-likes (cluster 1), fish prion proteins (cluster 2), tetrapode prion proteins (cluster 3) and doppels (cluster 4). We showed that the entire prion protein conformationally plastic region is well conserved between eutherian prion proteins and shadoos (18–25% identity and 28–34% similarity), and there could be a potential structural compatibility between shadoos and the left-handed parallel beta-helical fold. It is likely that the conserved genomic elements identified in this analysis represent bona fide cis-elements. However, this idea needs to be confirmed by functional assays in transgenic systems.

A new paradigm for developmental biology.

Abstract: It is usually thought that the development of complex organisms is controlled by protein regulatory factors and morphogenetic signals exchanged between cells and differentiating tissues during ontogeny. However, it is now evident that the majority of all animal genomes is transcribed, apparently in a developmentally regulated manner, suggesting that these genomes largely encode RNA machines and that there may be a vast hidden layer of RNA regulatory transactions in the background. I propose that the epigenetic trajectories of differentiation and development are primarily programmed by feed-forward RNA regulatory networks and that most of the information required for multicellular development is embedded in these networks, with cell-cell signalling required to provide important positional information and to correct stochastic errors in the endogenous RNA-directed program.

Thiamine biosynthesis in algae is regulated by riboswitches.

Abstract: In bacteria, many genes involved in the biosynthesis of cofactors such as thiamine pyrophosphate (TPP) are regulated by ribo switches, regions in the 5′ end of mRNAs to which the cofactor binds, thereby affecting translation and/or transcription. TPP riboswitches have now been identified in fungi, in which they alter mRNA splicing. Here, we show that addition of thiamine to cultures of the model green alga Chlamydomonas reinhardtii alters splicing of transcripts for the THI4 and THIC genes, encoding the first enzymes of the thiazole and pyrimidine branches of thiamine biosynthesis, respectively, concomitant with an increase in intracellular thiamine and TPP levels. Comparison with Volvox carteri, a related alga, revealed highly conserved regions within introns of these genes. Inspection of the sequences identified TPP riboswitch motifs, and RNA transcribed from the regions binds TPP in vitro. The THI4 riboswitch, but not the promoter region, was found to be necessary and sufficient for thiamine to repress expression of a luciferase-encoding reporter construct in vivo. The pyr1 mutant of C. reinhardtii, which is resistant to the thiamine analogue pyrithiamine, has a mutation in the THI4 riboswitch that prevents the THI4 gene from being repressed by TPP. By the use of these ribo switches, thiamine biosynthesis in C. reinhardtii can be effectively regulated at physiological concentrations of the vitamin.

Improperly Terminated, Unpolyadenylated mRNA of Sense Transgenes Is Targeted by RDR6-Mediated RNA Silencing in Arabidopsis

Abstract: RNA silencing can be induced by highly transcribed transgenes through a pathway dependent on RNA-DEPENDENT RNA POLYMERASE6 (RDR6) and may function as a genome protection mechanism against excessively expressed genes. Whether all transcripts or just aberrant transcripts activate this protection mechanism is unclear. Consistent RNA silencing induced by a transgene with three direct repeats of the β-glucuronidase (GUS) open reading frame (ORF) is associated with high levels of truncated, unpolyadenylated transcripts, probably from abortive transcription elongation. Truncated, unpolyadenylated transcripts from triple GUS ORF repeats were degraded in the wild type but accumulated in an rdr6 mutant, suggesting targeting for degradation by RDR6-mediated RNA silencing. A GUS transgene without a 3′ transcription terminator produced unpolyadenylated readthrough mRNA and consistent RDR6-dependent RNA silencing. Both GUS triple repeats and terminator-less GUS transgenes silenced an expressed GUS transgene in trans in the wild type but not in the rdr6 mutant. Placing two 3′ terminators in the GUS transgene 3′ reduced mRNA 3′ readthrough, decreased GUS-specific small interfering RNA accumulation, and enhanced GUS gene expression. Moreover, RDR6 was localized in the nucleus. We propose that improperly terminated, unpolyadenylated mRNA from transgene transcription is subject to RDR6-mediated RNA silencing, probably by acting as templates for the RNA polymerase, in Arabidopsis thaliana.

Genome mapping and expression analyses of human intronic noncoding RNAs reveal tissue-specific patterns and enrichment in genes related to regulation of transcription

Abstract: Background RNAs transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression. However, the complement of human genes in which introns are transcribed, and the number of intronic transcriptional units and their tissue expression patterns are not known.

Pre-mRNA Secondary Structures Influence Exon Recognition

Abstract: The secondary structure of a pre-mRNA influences a number of processing steps including alternative splicing. Since most splicing regulatory proteins bind to single-stranded RNA, the sequestration of RNA into double strands could prevent their binding. Here, we analyzed the secondary structure context of experimentally determined splicing enhancer and silencer motifs in their natural pre-mRNA context. We found that these splicing motifs are significantly more single-stranded than controls. These findings were validated by transfection experiments, where the effect of enhancer or silencer motifs on exon skipping was much more pronounced in single-stranded conformation. We also found that the structural context of predicted splicing motifs is under selection, suggesting a general importance of secondary structures on splicing and adding another level of evolutionary constraints on pre-mRNAs. Our results explain the action of mutations that affect splicing and indicate that the structural context of splicing motifs is part of the mRNA splicing code.

Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs

Abstract: The MBNL and CELF proteins act antagonistically to control the alternative splicing of specific exons during mammalian postnatal development. This process is dysregulated in myotonic dystrophy because MBNL proteins are sequestered by (CUG)n and (CCUG)n RNAs expressed from mutant DMPK and ZNF9 genes, respectively. While these observations predict that MBNL proteins have a higher affinity for these pathogenic RNAs versus their normal splicing targets, we demonstrate that MBNL1 possesses comparably high affinities for (CUG)n and (CAG)n RNAs as well as a splicing target, Tnnt3. Mapping of a MBNL1-binding site upstream of the Tnnt3 fetal exon indicates that a preferred binding site for this protein is a GC-rich RNA hairpin containing a pyrimidine mismatch. To investigate how pathogenic RNAs sequester MBNL1 in DM1 cells, we used a combination of chemical/enzymatic structure probing and electron microscopy to determine that MBNL1 forms a ring-like structure which binds to the dsCUG helix. While the MBNL1 N-terminal region is required for RNA binding, the C-terminal region mediates homotypic interactions which may stabilize intra- and/or inter-ring interactions. Our results provide a mechanistic basis for dsCUG-induced MBNL1 sequestration and highlight a striking similarity in the binding sites for MBNL proteins on splicing precursor and pathogenic RNAs.