http://genome.cshlp.org/content/3/5/272.abstract?sid=3910ac8d-5e4c-46f2-b7ef-d374fc02a5e9
Thursday, December 24, 2009
RNA associated with a heterodimeric protein that activates a meiotic homologous recombination hot spot: RL/RT/PCR strategy for cloning any unknown RNA
The ade6-M26 mutation in the fission yeast Schizosaccharomyces pombe creates a meiotic homologous recombination hot spot. We have achieved 40,000-fold purification of a heterodimeric DNA-binding protein, Mts1/Mts2, that activates the recombination hot spot. Physical studies suggested the presence of a third subunit. It is demonstrated here that RNA molecules of approximately 210 nucleotides copurified with the heterodimer. To characterize the RNA component, it was necessary to develop a new strategy for cloning of the unknown, low-abundance, partially degraded RNAs that were present in purified Mts1/Mts2 protein preparations. The strategy uses RNA ligase to add DNA oligonucleotide priming sites to the RNA for subsequent reverse transcription and PCR (RNA ligase, reverse transcription-PCR, or RL/RT/PCR). This cloning procedure could be applied to the cloning of any unknown RNA or DNA molecules. Because the cDNA clones obtained from Mts1/Mts2 were largely heterogeneous, it seems likely that the RNAs copurified as a result of tight but nonspecific interactions with the heterodimeric protein.
MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant
The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is ∼155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1–7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs.
http://genome.cshlp.org/content/16/10/1276.abstract?sid=3910ac8d-5e4c-46f2-b7ef-d374fc02a5e9
Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription
Pre-mRNA processing often occurs in coordination with transcription thereby coupling these two key regulatory events. As such, many proteins involved in mRNA processing associate with the transcriptional machinery and are in proximity to DNA. This proximity allows for the mapping of the genomic associations of RNA binding proteins by chromatin immunoprecipitation (ChIP) as a way of determining their sites of action on the encoded mRNA. Here, we used ChIP combined with high-density microarrays to localize on the human genome three functionally distinct RNA binding proteins: the splicing factor polypyrimidine tract binding protein (PTBP1/hnRNP I), the mRNA export factor THO complex subunit 4 (ALY/THOC4), and the 3′ end cleavage stimulation factor 64 kDa (CSTF2). We observed interactions at promoters, internal exons, and 3′ ends of active genes. PTBP1 had biases toward promoters and often coincided with RNA polymerase II (RNA Pol II). The 3′ processing factor, CSTF2, had biases toward 3′ ends but was also observed at promoters. The mRNA processing and export factor, ALY, mapped to some exons but predominantly localized to introns and did not coincide with RNA Pol II. Because the RNA binding proteins did not consistently coincide with RNA Pol II, the data support a processing mechanism driven by reorganization of transcription complexes as opposed to a scanning mechanism. In sum, we present the mapping in mammalian cells of RNA binding proteins across a portion of the genome that provides insight into the transcriptional assembly of RNA–protein complexes.
http://genome.cshlp.org/content/16/7/912.abstract?sid=3910ac8d-5e4c-46f2-b7ef-d374fc02a5e9
Comparative genomics beyond sequence-based alignments: RNA structures in the ENCODE regions
structure directly in any searches for these elements.
http://genome.cshlp.org/content/18/2/242.abstract?sid=3910ac8d-5e4c-46f2-b7ef-d374fc02a5e9
Detection of HCV RNA by the asymmetric gap ligase chain reaction.
The ligase chain reaction (LCR) and the gap ligase chain reaction (gLCR) are exponential amplification techniques for the detection of DNA sequences in a sample. Both techniques depend on the enzyme, DNA ligase, to join adjacent probes annealed to a DNA molecule. However, DNA ligase joins DNA inefficiency on an RNA target. Consequently, LCR and gLCR cannot amplify RNA efficiency. RNA detection methods using LCR or gLCR require a cDNA synthesis step. The carryover of four dNTPs from the cDNA reaction inhibits gLCR. Although LCR can use cDNA reaction products directly, background generated by blunt-end ligation does not allow the high sensitivity typically needed for HIV or HCV detection. The asymmetric gap ligase chain reaction (AGLCR) is a modification of gLCR that allows for the detection of RNA by using < or =" 3" class="search-term-highlight">RNA transcript can be reproducibly detected. HCV, an RNA virus with no DNA intermediate, was chosen as the initial RNA model system. HCV antibody-positive and normal samples were analyzed, and the results were found to correlate with the results obtained using nested RNA-PCR. AGLCR provides a new nucleic acid amplification technique that can aid in the diagnosis of disease when the detection of RNA is critical.
http://genome.cshlp.org/content/4/2/80.abstract?sid=3910ac8d-5e4c-46f2-b7ef-d374fc02a5e9
The evolution of genome compression and genomic novelty in RNA viruses
The genomes of RNA viruses are characterized by their extremely small size and extremely high mutation rates (typically 10 kb and 10−4/base/replication cycle, respectively), traits that are thought to be causally linked. One aspect of their small size is the genome compression caused by the use of overlapping genes (where some nucleotides code for two genes). Using a comparative analysis of all known RNA viral species, we show that viruses with larger genomes tend to have less gene overlap. We provide a numerical model to show how a high mutation rate could lead to gene overlap, and we discuss the factors that might explain the observed relationship between gene overlap and genome size. We also propose a model for the evolution of gene overlap based on the co-opting of previously unused ORFs, which gives rise to two types of overlap: (1) the creation of novel genes inside older genes, predominantly via +1 frameshifts, and (2) the incremental increase in overlap between originally contiguous genes, with no frameshift preference. Both types of overlap are viewed as the creation of genomic novelty under pressure for genome compression. Simulations based on our model generate the empirical size distributions of overlaps and explain the observed frameshift preferences. We suggest that RNA viruses are a good model system for the investigation of general evolutionary relationship between genome attributes such as mutational robustness, mutation rate, and size.
Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs
Long transcripts that do not encode protein have only rarely been the subject of experimental scrutiny. Presumably, this is owing to the current lack of evidence of their functionality, thereby leaving an impression that, instead, they represent “transcriptional noise.” Here, we describe an analysis of 3122 long and full-length, noncoding RNAs (“macroRNAs”) from the mouse, and compare their sequences and their promoters with orthologous sequence from human and from rat. We considered three independent signatures of purifying selection related to substitutions, sequence insertions and deletions, and splicing. We find that the evolution of the set of noncoding RNAs is not consistent with neutralist explanations. Rather, our results indicate that purifying selection has acted on the macroRNAs’ promoters, primary sequence, and consensus splice site motifs. Promoters have experienced the greatest elimination of nucleotide substitutions, insertions, and deletions. The proportion of conserved sequence (4.1%–5.5%) in these macroRNAs is comparable to the density of exons within protein-coding transcripts (5.2%). These macroRNAs, taken together, thus possess the imprint of purifying selection, thereby indicating their functionality. Our findings should now provide an incentive for the experimental investigation of these macroRNAs’ functions.
http://genome.cshlp.org/content/17/5/556.abstract?sid=3910ac8d-5e4c-46f2-b7ef-d374fc02a5e9
An improved method for the detection of hepatitis C virus RNA in plasma utilizing heminested primers and internal control RNA.
The majority of transfusion-associated, non-A, non-B hepatitis cases are caused by hepatitis C virus (HCV), a positive-stranded RNA virus. Although high titers of HCV in clinical specimens have been reported, in some cases extremely low titers of virus are not uncommon. Therefore, an extremely sensitive and reliable assay is required to determine viremia and replication of HCV accurately. We report here the systematic investigation of factors influencing the detection of HCV RNA by a reverse transcription-polymerase chain reaction (RT-PCR) assay utilizing "drop in-drop out" heminested primers derived from the conserved 5' non-coding region of the viral genome. A genetically engineered 5' noncoding region has been constructed and used as an internal control. Addition of the control RNA to each test not only allowed semiquantitation of positive reactions but also validated the performance of reverse transcription and PCR for every specimen. The optimized heminested PCR (HN-PCR) protocol is capable of amplifying one molecule of cloned HCV DNA or 10 molecules of in vitro-transcribed HCV RNA to levels detectable in ethidium bromide-stained agarose gels. We evaluated the improved method for the detection of HCV RNA on a human plasma sample containing the pedigreed strain H of HCV with a chimpanzee infectious dose of 10(6)/ml. Utilizing the internal control RNA, we calculated 2 x 10(7) virions in 1 ml of the original human plasma. The HN-PCR achieves the sensitivity and specificity of the double-nested PCR (DN-PCR) in a simplified format that avoids the false-positive results associated with DN-PCR.
Source -
http://genome.cshlp.org/content/2/3/241.abstract?sid=3910ac8d-5e4c-46f2-b7ef-d374fc02a5e9
Tuesday, December 15, 2009
Gene Expression and RNA Splicing
The regulation of gene expression is a ubiquitous phenomenon and is involved in virtually every process central to an organism, ranging from the fertilization of germ cells, across the cell cycle, to stimuli–response pathways or apoptosis. To control the expression of genes under such diverse contexts, regulation occurs on different cellular levels and involves a series of complex biochemical mechanisms that one can broadly classify into transcription, RNA processing and cytoplasmic transport, and post-transcriptional control and translation. While a series of distinct machineries is involved in controlling gene expression at each level, these complex circuits bear signs of interconnectedness
In higher eukaryotes, splicing constitutes a critical mode for the regulation of gene expression at the level of RNA processing. The large majority of eukaryotic protein-coding genes are transcribed as precursors of messenger RNAs (pre-mRNAs), in which exons are separated from each other by intervening regions of non-protein–coding information (introns), which have to be correctly spliced out to produce a mature mRNA. Splicing of pre-mRNAs occurs in a two-step reaction. In the first step, the message is cleaved at the 5′ end of an intron, and this 5′ end is linked to the branch point, which is typically in close proximity upstream of the 3′ end of the intron. In the second step, the mRNA intermediate is cleaved at the 3′ splice site (3′ss), exons are ligated, and the intron lariat is released. During later stages of spliceosome assembly, the 5′ss and 3′ss pair and interact (typically across the exon, but pairing across an intron can occur), supported by general and specific splicing factors that recognize them. Typical mammalian genes span tens of thousands of nucleotides, with on average nine exons and protein-coding regions on the order of a thousand nucleotides, thus embedding “exon islands” within a large “sea” of noncoding nucleotides that have to be accurately recognized for correct splicing and exon ligation. This important task is executed in the nucleus by the spliceosome, a large ribonucleoprotein (RNP) complex that involves five small nuclear RNAs and potentially hundreds of proteins, the core components of which are highly conserved across metazoan genomes
Genome-wide mapping of alternative splicing
Alternative splicing can enhance transcriptome plasticity and proteome diversity. In plants, alternative splicing can be manifested at different developmental stages, and is frequently associated with specific tissue types or environmental conditions such as abiotic stress. We mapped the Arabidopsis transcriptome at single-base resolution using the Illumina platform for ultrahigh-throughput RNA sequencing (RNA-seq). Deep transcriptome sequencing confirmed a majority of annotated introns and identified thousands of novel alternatively spliced mRNA isoforms. Our analysis suggests that at least ∼42% of intron-containing genes in Arabidopsis are alternatively spliced; this is significantly higher than previous estimates based on cDNA/expressed sequence tag sequencing. Random validation confirmed that novel splice isoforms empirically predicted by RNA-seq can be detected in vivo. Novel introns detected by RNA-seq were substantially enriched in nonconsensus terminal dinucleotide splice signals. Alternative isoforms with premature termination codons (PTCs) comprised the majority of alternatively spliced transcripts. Using an example of an essential circadian clock gene, we show that intron retention can generate relatively abundant PTC+ isoforms and that this specific event is highly conserved among diverse plant species. Alternatively spliced PTC+ isoforms can be potentially targeted for degradation by the nonsense mediated mRNA decay (NMD) surveillance machinery or regulate the level of functional transcripts by the mechanism of regulated unproductive splicing and translation (RUST). We demonstrate that the relative ratios of the PTC+ and reference isoforms for several key regulatory genes can be considerably shifted under abiotic stress treatments. Taken together, our results suggest that like in animals, NMD and RUST may be widespread in plants and may play important roles in regulating gene expression.
Source - http://genome.cshlp.org/content/early/2009/11/18/gr.093302.109.abstract
Splice Site Analysis Tool Analysis
In-silico splice site prediction tools can be used to predict the effect of a genetic variant on splicing. A large number of prediction tools are currently available but only small scale analyses studies of these algorithms have been carried out. The UV guidelines provided by the CMGS suggest several splice site prediction algorithms, but the performance of these algorithms have not previously been formally assessed and may give divergent results. The splice site tool analysis performed by NGRL Manchester aims to provide a reliable assessment of the performance of these algorithms in the prediction of splicing-related variant pathogenicity. It also assesses the scope of the splice-site prediction tools to ensure that they can be used in the most appropriate way and the report shows scientists how to use splice site prediction tools in the prediction of pathogenesis with more confidence.
The report describes the assessment of six of the most common donor and acceptor prediction algorithms for their ability to predict the pathogenicity of splice site variants. In each algorithm the splice signal given by the wild type sequence is compared to the splice site signal given by a mutated sequence supplied by the user.
We conclude that the four algorithms used in Alamut were shown to have a high degree of accuracy and users can be confident in the safe interpretation of these results, while only one of the four algorithms cannot be used though a standalone web interface.
The range of splice site signal strength predictions given by the algorithms was determined by the position of the variant. Variants found between +7 and -10 from the splice site junction show a reduction in splicing predicted by the algorithms and it is in this range that the algorithms are likely to be the most useful.
Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere
The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. Here, we demonstrate that centromeric α-satellite RNA and proteins CENPC1 and INCENP accumulate in the human interphase nucleolus in an RNA polymerase I–dependent manner. The nucleolar targeting of CENPC1 and INCENP requires α-satellite RNA, as evident from the delocalization of both proteins from the nucleolus in RNase-treated cells, and the nucleolar relocalization of these proteins following α-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, we have identified the nucleolar localization sequences on CENPC1 and INCENP. We present evidence that CENPC1 is an RNA-associating protein that binds α-satellite RNA by an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and “RNA replenishment” experiments, we show that α-satellite RNA is a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere. Our data suggest that centromere satellite RNA directly facilitates the accumulation and assembly of centromere-specific nucleoprotein components at the nucleolus and mitotic centromere, and that the sequestration of these components in the interphase nucleolus provides a regulatory mechanism for their timely release into the nucleoplasm for kinetochore assembly at the onset of mitosis.
Source - http://genome.cshlp.org/content/17/8/1146.abstract?sid=4be18750-d5af-4896-b05e-eeb49c944425
Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription
Pre-mRNA processing often occurs in coordination with transcription thereby coupling these two key regulatory events. As such, many proteins involved in mRNA processing associate with the transcriptional machinery and are in proximity to DNA. This proximity allows for the mapping of the genomic associations of RNA binding proteins by chromatin immunoprecipitation (ChIP) as a way of determining their sites of action on the encoded mRNA. Here, we used ChIP combined with high-density microarrays to localize on the human genome three functionally distinct RNA binding proteins: the splicing factor polypyrimidine tract binding protein (PTBP1/hnRNP I), the mRNA export factor THO complex subunit 4 (ALY/THOC4), and the 3′ end cleavage stimulation factor 64 kDa (CSTF2). We observed interactions at promoters, internal exons, and 3′ ends of active genes. PTBP1 had biases toward promoters and often coincided with RNA polymerase II (RNA Pol II). The 3′ processing factor, CSTF2, had biases toward 3′ ends but was also observed at promoters. The mRNA processing and export factor, ALY, mapped to some exons but predominantly localized to introns and did not coincide with RNA Pol II. Because the RNA binding proteins did not consistently coincide with RNA Pol II, the data support a processing mechanism driven by reorganization of transcription complexes as opposed to a scanning mechanism. In sum, we present the mapping in mammalian cells of RNA binding proteins across a portion of the genome that provides insight into the transcriptional assembly of RNA–protein complexes.
Source - http://genome.cshlp.org/content/16/7/912.abstract?sid=4be18750-d5af-4896-b05e-eeb49c944425
The Largest Subunit of Human RNA Polymerase III Is Closely Related to the Largest Subunit of Yeast and Trypanosome RNA Polymerase III
In both yeast and mammalian systems, considerable progress has been made toward the characterization of the transcription factors required for transcription by RNA polymerase III. However, whereas in yeast all of the RNA polymerase III subunits have been cloned, relatively little is known about the enzyme itself in higher eukaryotes. For example, no higher eukaryotic sequence corresponding to the largest RNA polymerase III subunit is available. Here we describe the isolation of cDNAs that encode the largest subunit of human RNA polymerase III, as suggested by the observations that (1) antibodies directed against the cloned protein immunoprecipitate an active enzyme whose sensitivity to different concentrations of α-amanitin is that expected for human RNA polymerase III; and (2) depletion of transcription extracts with the same antibodies results in inhibition of transcription from an RNA polymerase III, but not from an RNA polymerase II, promoter. Sequence comparisons reveal that regions conserved in the RNA polymerase I, II, and III largest subunits characterized so far are also conserved in the human RNA polymerase III sequence, and thus probably perform similar functions for the human RNA polymerase III enzyme.
Source - http://genome.cshlp.org/content/7/10/1006.abstract?sid=4be18750-d5af-4896-b05e-eeb49c944425
Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa
The diversity of microRNAs and small-interfering RNAs has been extensively explored within angiosperms by focusing on a few key organisms such as Oryza sativa and Arabidopsis thaliana. A deeper division of the plants is defined by the radiation of the angiosperms and gymnosperms, with the latter comprising the commercially important conifers. The conifers are expected to provide important information regarding the evolution of highly conserved small regulatory RNAs. Deep sequencing provides the means to characterize and quantitatively profile small RNAs in understudied organisms such as these. Pyrosequencing of small RNAs from O. sativa revealed, as expected, ∼21- and ∼24-nt RNAs. The former contained known microRNAs, and the latter largely comprised intergenic-derived sequences likely representing heterochromatin siRNAs. In contrast, sequences from Pinus contorta were dominated by 21-nt small RNAs. Using a novel sequence-based clustering algorithm, we identified sequences belonging to 18 highly conserved microRNA families in P. contorta as well as numerous clusters of conserved small RNAs of unknown function. Using multiple methods, including expressed sequence folding and machine learning algorithms, we found a further 53 candidate novel microRNA families, 51 appearing specific to the P. contorta library. In addition, alignment of small RNA sequences to the O. sativa genome revealed six perfectly conserved classes of small RNA that included chloroplast transcripts and specific types of genomic repeats. The conservation of microRNAs and other small RNAs between the conifers and the angiosperms indicates that important RNA silencing processes were highly developed in the earliest spermatophytes. Genomic mapping of all sequences to the O. sativa genome can be viewed at http://microrna.bcgsc.ca/cgi-bin/gbrowse/rice_build_3/.
Source - http://genome.cshlp.org/content/18/4/571.abstract?sid=4be18750-d5af-4896-b05e-eeb49c944425
Clusters and superclusters of phased small RNAs in the developing inflorescence of rice
To address the role of small regulatory RNAs in rice development, we generated a large data set of small RNAs from mature leaves and developing roots, shoots, and inflorescences. Using a spatial clustering algorithm, we identified 36,780 genomic groups of small RNAs. Most consisted of 24-nt RNAs that are expressed in all four tissues and enriched in repeat regions of the genome; 1029 clusters were composed primarily of 21-nt small RNAs and, strikingly, 831 of these contained phased RNAs and were preferentially expressed in developing inflorescences. Thirty-eight of the 24-mer clusters were also phased and preferentially expressed in inflorescences. The phased 21-mer clusters derive from nonprotein coding, nonrepeat regions of the genome and are grouped together into superclusters containing 10–46 clusters. The majority of these 21-mer clusters (705/831) are flanked by a degenerate 22-nt motif that is offset by 12 nt from the main phase of the cluster. Small RNAs complementary to these flanking 22-nt motifs define a new miRNA family, which is conserved in maize and expressed in developing reproductive tissues in both plants. These results suggest that the biogenesis of phased inflorescence RNAs resembles that of tasiRNAs and raise the possibility that these novel small RNAs function in early reproductive development in rice and other monocots.
Source - http://genome.cshlp.org/content/19/8/1429.abstract?sid=4be18750-d5af-4896-b05e-eeb49c944425
MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant
The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is ∼155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1–7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs.
Source - http://genome.cshlp.org/content/16/10/1276.abstract?sid=4be18750-d5af-4896-b05e-eeb49c944425
A survey of RNA editing in human brain
We have conducted a survey of RNA editing in human brain by comparing sequences of clones from a human brain cDNA library to the reference human genome sequence and to genomic DNA from the same individual. In the RNA sample from which the library was constructed, ∼1:2000 nucleotides were edited out of >3 Mb surveyed. All edits were adenosine to inosine (A→I) and were predominantly in intronic and in intergenic RNAs. No edits were found in translated exons and few in untranslated exons. Most edits were in high-copy-number repeats, usually Alus. Analysis of the genome in the vicinity of edited sequences strongly supports the idea that formation of intramolecular double-stranded RNA with an inverted copy underlies most A→I editing. The likelihood of editing is increased by the presence of two inverted copies of a sequence within the same intron, proximity of the two sequences to each other (preferably within 2 kb), and by a high density of inverted copies in the vicinity. Editing exhibits sequence preferences and is less likely at an adenosine 3′ to a guanosine and more likely at an adenosine 5′ to a guanosine. Simulation by BLAST alignment of the double-stranded RNA molecules that underlie known edits indicates that there is a greater likelihood of A→I editing at A:C mismatches than editing at other mismatches or at A:U matches. However, because A:U matches in double-stranded RNA are more common than all mismatches, overall the likely effect of editing is to increase the number of mismatches in double-stranded RNA.
Source - http://genome.cshlp.org/content/14/12/2379.abstract?sid=4be18750-d5af-4896-b05e-eeb49c944425
Abundant and dynamically expressed miRNAs, piRNAs, and other small RNAs in the vertebrate Xenopus tropicalis
Source - http://genome.cshlp.org/content/19/10/1766.abstract?sid=4be18750-d5af-4896-b05e-eeb49c944425
Analysis of the 5S RNA Pool in Arabidopsis thaliana: RNAs Are Heterogeneous and Only Two of the Genomic 5S Loci Produce Mature 5S RNA
Source - http://genome.cshlp.org/content/12/1/132.abstract?sid=4be18750-d5af-4896-b05e-eeb49c944425
RNA associated with a heterodimeric protein that activates a meiotic homologous recombination hot spot: RL/RT/PCR strategy for cloning any unknown RNA
The ade6-M26 mutation in the fission yeast Schizosaccharomyces pombe creates a meiotic homologous recombination hot spot. We have achieved 40,000-fold purification of a heterodimeric DNA-binding protein, Mts1/Mts2, that activates the recombination hot spot. Physical studies suggested the presence of a third subunit. It is demonstrated here that RNA molecules of approximately 210 nucleotides copurified with the heterodimer. To characterize the RNA component, it was necessary to develop a new strategy for cloning of the unknown, low-abundance, partially degraded RNAs that were present in purified Mts1/Mts2 protein preparations. The strategy uses RNA ligase to add DNA oligonucleotide priming sites to the RNA for subsequent reverse transcription and PCR (RNA ligase, reverse transcription-PCR, or RL/RT/PCR). This cloning procedure could be applied to the cloning of any unknown RNA or DNA molecules. Because the cDNA clones obtained from Mts1/Mts2 were largely heterogeneous, it seems likely that the RNAs copurified as a result of tight but nonspecific interactions with the heterodimeric protein.
Source - http://genome.cshlp.org/content/3/5/272.abstract?sid=4be18750-d5af-4896-b05e-eeb49c944425
Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts
Metazoan genes are encrypted with at least two superimposed codes: the genetic code to specify the primary structure of proteins and the splicing code to expand their proteomic output via alternative splicing. Here, we define the specificity of a central regulator of pre-mRNA splicing, the conserved, essential splicing factor SFRS1. Cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) identified 23,632 binding sites for SFRS1 in the transcriptome of cultured human embryonic kidney cells. SFRS1 was found to engage many different classes of functionally distinct transcripts including mRNA, miRNA, snoRNAs, ncRNAs, and conserved intergenic transcripts of unknown function. The majority of these diverse transcripts share a purine-rich consensus motif corresponding to the canonical SFRS1 binding site. The consensus site was not only enriched in exons cross-linked to SFRS1 in vivo, but was also enriched in close proximity to splice sites. mRNAs encoding RNA processing factors were significantly overrepresented, suggesting that SFRS1 may broadly influence the post-transcriptional control of gene expression in vivo. Finally, a search for the SFRS1 consensus motif within the Human Gene Mutation Database identified 181 mutations in 82 different genes that disrupt predicted SFRS1 binding sites. This comprehensive analysis substantially expands the known roles of human SR proteins in the regulation of a diverse array of RNA transcripts.
Source - http://genome.cshlp.org/content/19/3/381.abstract?sid=40206306-7c2e-4b18-96b3-8319fa594a4b
A genomic analysis of RNA polymerase II modification and chromatin architecture related to 3′ end RNA polyadenylation
Genomic analyses have been applied extensively to analyze the process of transcription initiation in mammalian cells, but less to transcript 3′ end formation and transcription termination. We used a novel approach to prepare 3′ end fragments from polyadenylated RNA, and mapped the position of the poly(A) addition site using oligonucleotide arrays tiling 1% of the human genome. This approach revealed more 3′ ends than had been annotated. The distribution of these ends relative to RNA polymerase II (PolII) and di- and trimethylated lysine 4 and lysine 36 of histone H3 was compared. A substantial fraction of unannotated 3′ ends of RNA are intronic and antisense to the embedding gene. Poly(A) ends of annotated messages lie on average 2 kb upstream of the end of PolII binding (termination). Near the termination sites, and in some internal sites, unphosphorylated and C-terminal domain (CTD) serine 2 phosphorylated PolII (POLR2A) accumulate, suggesting pausing of the polymerase and perhaps dephosphorylation prior to release. Lysine 36 trimethylation occurs across transcribed genes, sometimes alternating with stretches of DNA in which lysine 36 dimethylation is more prominent. Lysine 36 methylation decreases at or near the site of polyadenylation, sometimes disappearing before disappearance of phosphorylated RNA PolII or release of PolII from DNA. Our results suggest that transcription termination loss of histone 3 lysine 36 methylation and later release of RNA polymerase. The latter is often associated with polymerase pausing. Overall, our study reveals extensive sites of poly(A) addition and provides insights into the events that occur during 3′ end formation.
Source - http://genome.cshlp.org/content/18/8/1224.abstract?sid=40206306-7c2e-4b18-96b3-8319fa594a4b
Genome-wide discovery and verification of novel structured RNAs in Plasmodium falciparum
Source - http://genome.cshlp.org/content/18/2/281.abstract?sid=40206306-7c2e-4b18-96b3-8319fa594a4b
Simultaneous detection of DNA and RNA by differential polymerase chain reaction (DIFF-PCR).
A new technique, the differential polymerase chain reaction (DIFF-PCR), allows the simultaneous amplification of DNA and homologous RNA in a single assay by the combination of DNA-PCR and RNA-PCR on the same target. DNA-PCR amplifies a selected segment of dsDNA, whereas RNA-PCR amplifies a complementary DNA (cDNA), produced by reverse transcription of RNA. In a mixture of target DNA and RNA, DNA is amplified using a combination of sense and antisense primers under high-stringency conditions giving a D-amplicon. RNA is first reverse-transcribed with a primer carrying a nontarget 5' end into a tagged cDNA at low stringency. Tagged cDNA is subsequently amplified, providing an R-amplicon smaller in size than the D-amplicon. By quantifying the relative amounts of amplified RNA and homologous DNA, a sensitive measure for the transcription rate of a defined DNA segment is obtained. Thus, DIFF-PCR may serve as a useful tool for monitoring gene expression as well as for studying gene regulation and gene function.
Source - http://genome.cshlp.org/content/3/1/23.abstract?sid=40206306-7c2e-4b18-96b3-8319fa594a4b
Selective RNA amplification: a novel method using dUMP-containing primers and uracil DNA glycosylase.
The application of PCR to a wide variety of biological problems and molecular techniques has gained wide acceptance. RNA-PCR, a technique in which first-strand cDNA synthesis is followed by PCR amplification, has enabled detection and characterization of rare transcripts. One problem confronting the researcher involves specific amplification of transcribed sequences in the presence of small amounts of genomic DNA of identical sequence. We describe a novel technique, selective RNA amplification, which will specifically amplify RNA sequences in a background of homologous DNA. The method involves first-strand cDNA synthesis from a specific dUMP-containing oligonucleotide that contains unique user-defined 5' sequence (adapter sequence) not found in the message of interest. RNA template is degraded using RNase H, which is specific for RNA/DNA hybrids. This is followed by second-strand synthesis using a gene-specific primer (GSP). The original adapter primer is digested with uracil DNA glycosylase (UDG) to prevent its participation in subsequent amplification. PCR is then performed using the GSP and a second primer corresponding to the unique adapter sequence. In this paper, we apply this method to the amplification of RNA derived from human papilloma virus sequences. Using Southern analysis, we demonstrate specific amplification of 10(5) molecules of an in vitro-transcribed RNA. Denatured DNA of identical sequence and concentration was not amplified using the RNA-specific method. The method could eliminate the need for stringent purification of RNA and enables amplification of rare messages from RNA preparations containing homologous DNA of identical sequence and size.
Source - http://genome.cshlp.org/content/3/1/28.abstract?sid=40206306-7c2e-4b18-96b3-8319fa594a4b
An improved method for the detection of hepatitis C virus RNA in plasma utilizing heminested primers and internal control RNA.
Source - http://genome.cshlp.org/content/2/3/241.abstract?sid=40206306-7c2e-4b18-96b3-8319fa594a4b
Genome-wide identification and analysis of small RNAs originated from natural antisense transcripts in Oryza sativa
Natural antisense transcripts (NATs) have been shown to play important roles in post-transcriptional regulation through the RNA interference pathway. We have combined pyrophosphate-based high-throughput sequencing and computational analysis to identify and analyze, in genome scale, cis-NAT and trans-NAT small RNAs that are derived under normal conditions and in response to drought and salt stresses in the staple plant Oryza sativa. Computationally, we identified 344 cis-NATs and 7142 trans-NATs that are formed by protein-coding genes. From the deep sequencing data, we found 108 cis-NATs and 7141 trans-NATs that gave rise to small RNAs from their overlapping regions. Consistent with early findings, the majority of these 108 cis-NATs seem to be associated with specific conditions or developmental stages. Our analyses also revealed several interesting results. The overlapping regions of the cis-NATs and trans-NATs appear to be more enriched with small RNA loci than non-overlapping regions. The small RNAs generated from cis-NATs and trans-NATs have a length bias of 21 nt, even though their lengths spread over a large range. Furthermore, >40% of the small RNAs from cis-NATs and trans-NATs carry an A as their 5′-terminal nucleotides. A substantial portion of the transcripts are involved in both cis-NATs and trans-NATs, and many trans-NATs can form many-to-many relationships, indicating that NATs may form complex regulatory networks in O. sativa. This study is the first genome-wide investigation of NAT-derived small RNAs in O. sativa. It reveals the importance of NATs in biogenesis of small RNAs and broadens our understanding of the roles of NAT-derived small RNAs in gene regulation, particularly in response to environmental stimuli.
Source - http://genome.cshlp.org/content/19/1/70.abstract?sid=40206306-7c2e-4b18-96b3-8319fa594a4b
Detection of HCV RNA by the asymmetric gap ligase chain reaction.
The ligase chain reaction (LCR) and the gap ligase chain reaction (gLCR) are exponential amplification techniques for the detection of DNA sequences in a sample. Both techniques depend on the enzyme, DNA ligase, to join adjacent probes annealed to a DNA molecule. However, DNA ligase joins DNA inefficiency on an RNA target. Consequently, LCR and gLCR cannot amplify RNA efficiency. RNA detection methods using LCR or gLCR require a cDNA synthesis step. The carryover of four dNTPs from the cDNA reaction inhibits gLCR. Although LCR can use cDNA reaction products directly, background generated by blunt-end ligation does not allow the high sensitivity typically needed for HIV or HCV detection. The asymmetric gap ligase chain reaction (AGLCR) is a modification of gLCR that allows for the detection of RNA by using < or =" 3" class="search-term-highlight">RNA transcript can be reproducibly detected. HCV, an RNA virus with no DNA intermediate, was chosen as the initial RNA model system. HCV antibody-positive and normal samples were analyzed, and the results were found to correlate with the results obtained using nested RNA-PCR. AGLCR provides a new nucleic acid amplification technique that can aid in the diagnosis of disease when the detection of RNA is critical.
Source - http://genome.cshlp.org/content/4/2/80.abstract?sid=40206306-7c2e-4b18-96b3-8319fa594a4b
Mouse let-7 miRNA populations exhibit RNA editing that is constrained in the 5′-seed/ cleavage/anchor regions and stabilize predicted mmu-let-7a:mRNA
Massively parallel sequencing of millions of <30-nt class="search-term-highlight">RNAs expressed in mouse ovary, embryonic pancreas (E14.5), and insulin-secreting beta-cells (βTC-3) reveals that ∼50% of the mature miRNAs representing mostly the mmu-let-7 family display internal insertion/deletions and substitutions when compared to precursor miRNA and the mouse genome reference sequences. Approximately, 12%–20% of species associated with mmu-let-7 populations exhibit sequence discrepancies that are dramatically reduced in nucleotides 3–7 (5′-seed) and 10–15 (cleavage and anchor sites). This observation is inconsistent with sequencing error and leads us to propose that the changes arise predominantly from post-transcriptional RNA-editing activity operating on miRNA:target mRNA complexes. Internal nucleotide modifications are most enriched at the ninth nucleotide position. A common ninth base edit of U-to-G results in a significant increase in stability of down-regulated let-7a targets in inhibin-deficient mice (Inha−/−). An excess of U-insertions (14.8%) over U-deletions (1.5%) and the presence of cleaved intermediates suggest that a mammalian TUTase (terminal uridylyl transferase) mediated dUTP-dependent U-insertion/U-deletion cycle may be a possible mechanism. We speculate that mRNA target site-directed editing of mmu-let-7a duplex-bulges stabilizes “loose” miRNA:mRNA target associations and functions to expand the target repertoire and/or enhance mRNA decay over translational repression. Our results also demonstrate that the systematic study of sequence variation within specific RNA classes in a given cell type from millions of sequences generated by next-generation sequencing (NGS) technologies (“intranomics”) can be used broadly to infer functional constraints on specific parts of completely uncharacterized RNAs.
Source - http://genome.cshlp.org/content/18/10/1571.abstract?sid=40206306-7c2e-4b18-96b3-8319fa594a4b
Researchers unravel the mysteries of DNA packaging
Imagine a huge spool of film containing thousands of sequences of random scenes. Without a talented editor, a screening would have no meaning.
The RNA “spools” that make up DNA in our genes need careful editing, too. Genes are composed of meaningful sequences, called exons, separated by meaningless junk sections called introns. In order for cells to produce RNA — the material that is required to create proteins that are vital for life — they must precisely remove meaningless introns and bind meaningful exons together, a process called “splicing.”
How cells differentiate between what’s useful and what’s garbage in our complicated and messy genetic code is a fundamental biology question — one with extremely important implications. Now, Prof. Gil Ast and his doctoral student Schraga Schwartz at the Sackler School of Medicine at
Their groundbreaking findings, recently published in Nature Structural and Molecular Biology, reveal a new mechanism to explain how splicing works. They’ve discovered that the structure of DNA itself affects the ways RNA is spliced. “These findings,” says Prof. Ast, “will bring us closer to understanding diseases like cystic fibrosis and certain forms of cancer that result from our cells’ failure to edit sequences properly.”
Rewriting textbook science on DNA
Until now, how RNA was “edited” to fit together has been a mystery. The TAU revelations provide important information about creating proteins, and give new clues to drug developers to better understand how diseases such as cancer and genetic disorders operate at the gene level. That insight can offer significant new cellular mechanisms to create innovative drug therapies.
“We’ve found something previously unknown,” Prof. Ast explains. “At the DNA level, exons are packaged differently than introns. This fact is significant, telling us a process of gene expression is taking place at an earlier step than previously believed.” …
Source - http://xenophilius.wordpress.com/2009/12/15/researchers-unravel-the-mysteries-of-dna-packaging/
Subscribe to:
Posts (Atom)
