Endonucleolytic Cleavage Research Articles

Bacillus subtilis RNase Y Activity In Vivo Analysed by Tiling Microarrays

RNase Y is a key endoribonuclease affecting global mRNA stability in Bacillus subtilis. Its characterization provided the first evidence that endonucleolytic cleavage plays a major role in the mRNA metabolism of this organism. RNase Y shares important functional features with the RNA decay initiating RNase E from Escherichia coli, notably a similar cleavage specificity and a preference for 5′ monophosphorylated substrates. We used high-resolution tiling arrays to analyze the effect of RNase Y depletion on RNA abundance covering the entire genome. The data confirm that this endoribonuclease plays a key role in initiating the decay of a large number of mRNAs as well as non coding RNAs. The downstream cleavage products are likely to be degraded by the 5′ exonucleolytic activity of RNases J1/J2 as we show for a specific case. Comparison of the data with that of two other recent studies revealed very significant differences. About two thirds of the mRNAs upregulated following RNase Y depletion were different when compared to either one of these studies and only about 10% were in common in all three studies. This highlights that experimental conditions and data analysis play an important role in identifying RNase Y substrates by global transcriptional profiling. Our data confirmed already known RNase Y substrates and due to the precision and reproducibility of the profiles allow an exceptionally detailed view of the turnover of hundreds of new RNA substrates.

A global profiling of uncapped mRNAs under cold stress reveals specific decay patterns and endonucleolytic cleavages in Brachypodium distachyon

BackgroundmRNA degradation is a critical factor in determining mRNA abundance and enables rapid adjustment of gene expression in response to environmental stress. The involvement of processing bodies in stress response suggests a role for decapping-mediated mRNA degradation. However, little is known about the role of mRNA degradation under stressful environmental conditions.ResultsHere, we perform a global study of uncapped mRNAs, via parallel analysis of RNA ends (PARE), under cold stress in Brachypodium distachyon. Enrichment analysis indicates that degradation products detected by PARE are mainly generated by the decapping pathway. Endonucleolytic cleavages are detected, uncovering another way of modulating gene expression. PARE and RNA-Seq analyses identify four types of mRNA decay patterns. Type II genes, for which light-harvesting processes are over-represented in gene ontology analyses, show unchanged transcript abundance and altered uncapped transcript abundance. Uncapping-mediated transcript stability of light harvesting-related genes changes significantly in response to cold stress, which may allow rapid adjustments in photosynthetic activity in response to cold stress. Transcript abundance and uncapped transcript abundance for type III genes changes in opposite directions in response to cold stress, indicating that uncapping-mediated mRNA degradation plays a role in regulating gene expression.ConclusionTo our knowledge, this is the first global analysis of mRNA degradation under environmental stress conditions in Brachypodium distachyon. We uncover specific degradation and endonucleolytic cleavage patterns under cold stress, which will deepen our understanding of mRNA degradation under stressful environmental conditions, as well as the cold stress response mechanism in monocots.

A Complex Containing the CPSF73 Endonuclease and Other Polyadenylation Factors Associates with U7 snRNP and Is Recruited to Histone Pre-mRNA for 3′-End Processing

Animal replication-dependent histone pre-mRNAs are processed at the 3' end by endonucleolytic cleavage that is not followed by polyadenylation. The cleavage reaction is catalyzed by CPSF73 and depends on the U7 snRNP and its integral component, Lsm11. A critical role is also played by the 220-kDa protein FLASH, which interacts with Lsm11. Here we demonstrate that the N-terminal regions of these two proteins form a platform that tightly interacts with a unique combination of polyadenylation factors: symplekin, CstF64, and all CPSF subunits, including the endonuclease CPSF73. The interaction is inhibited by alterations in each component of the FLASH/Lsm11 complex, including point mutations in FLASH that are detrimental for processing. The same polyadenylation factors are associated with the endogenous U7 snRNP and are recruited in a U7-dependent manner to histone pre-mRNA. Collectively, our studies identify the molecular mechanism that recruits the CPSF73 endonuclease to histone pre-mRNAs, reveal an unexpected complexity of the U7 snRNP, and suggest that in animal cells polyadenylation factors assemble into two alternative complexes-one specifically crafted to generate polyadenylated mRNAs and the other to generate nonpolyadenylated histone mRNAs that end with the stem-loop.

PARP1 Represses PAP and Inhibits Polyadenylation during Heat Shock

The 3' ends of most eukaryotic mRNAs are produced by an endonucleolytic cleavage followed by synthesis of a poly(A) tail. Poly(A) polymerase (PAP), the enzyme that catalyzes the formation of the tail, is subject to tight regulation involving several posttranslational modifications. Here we show that the enzyme poly(ADP-ribose) polymerase 1 (PARP1) modifies PAP and regulates its activity both in vitro and in vivo. PARP1 binds to and modifies PAP by poly(ADP-ribosyl)ation (PARylation) in vitro, which inhibits PAP activity. In vivo we show that PAP is PARylated during heat shock, leading to inhibition of polyadenylation in a PARP1-dependent manner. The observed inhibition reflects reduced RNA binding affinity of PARylated PAP in vitro and decreased PAP association with non-heat shock protein-encoding genes in vivo. Our results provide direct evidence that PARylation can control processing of mRNA precursors, and also identify PARP1 as a regulator of polyadenylation during thermal stress.

Tick-box for 3'-end formation of mitochondrial transcripts in Ixodida, basal chelicerates and Drosophila.

According to the tRNA punctuation model, the mitochondrial genome (mtDNA) of mammals and arthropods is transcribed as large polycistronic precursors that are maturated by endonucleolytic cleavage at tRNA borders and RNA polyadenylation. Starting from the newly sequenced mtDNA of Ixodes ricinus and using a combination of mitogenomics and transcriptional analyses, we found that in all currently-sequenced tick lineages (Prostriata, Metastriata and Argasidae) the 3′-end of the polyadenylated nad1 and rrnL transcripts does not follow the tRNA punctuation model and is located upstream of a degenerate 17-bp DNA motif. A slightly different motif is also present downstream the 3′-end of nad1 transcripts in the primitive chelicerate Limulus polyphemus and in Drosophila species, indicating the ancient origin and the evolutionary conservation of this motif in arthropods. The transcriptional analyses suggest that this motif directs the 3′-end formation of the nad1/rrnL mature RNAs, likely working as a transcription termination signal or a processing signal of precursor transcripts. Moreover, as most regulatory elements, this motif is characterized by a taxon-specific evolution. Although this signal is not exclusive of ticks, making a play on words it has been named “Tick-Box”, since it is a check mark that has to be verified for the 3′-end formation of some mt transcripts, and its consensus sequence has been here carefully characterized in ticks. Indeed, in the whole mtDNA of all ticks, the Tick-Box is always present downstream of nad1 and rrnL, mainly in non-coding regions (NCRs) and occasionally within trnL(CUN). However, some metastriates present a third Tick-Box at an intriguing site - inside the small NCR located at one end of a 3.4 kb translocated region, the other end of which exhibits the nad1 Tick-Box - hinting that this motif could have been involved in metastriate gene order rearrangements.

Intronic hammerhead ribozymes in mRNA biogenesis

Small self-cleaving ribozymes are a group of natural RNAs that are capable of catalyzing their own and sequence-specific endonucleolytic cleavage. One of the most studied members is the hammerhead ribozyme (HHR), a catalytic RNA originally discovered in subviral plant pathogens but recently shown to reside in a myriad of genomes along the tree of life. In eukaryotes, most of the genomic HHRs seem to be related to short interspersed retroelements, with the main exception of a group of strikingly conserved ribozymes found in the genomes of all amniotes (reptiles, birds and mammals). These amniota HHRs occur in the introns of a few specific genes, and clearly point to a preserved biological role during pre-mRNA biosynthesis. More specifically, bioinformatic analysis suggests that these intronic ribozymes could offer a new form of splicing regulation of the mRNA of higher vertebrates. We review here the latest advances in the discovery and biological characterization of intronic HHRs of vertebrates, including new conserved examples in the genomes of the primitive turtle and coelacanth fish.

Backbone resonance assignments for a homolog of the essential ribosome biogenesis factor Fap7 from P. horikoshii in its nucleotide-free and -bound forms

The protein "factor activating Pos9 (Skn7)", Fap7, is an essential protein in yeast and plays an important role in the biogenesis of the small ribosomal subunit. In eukaryotes, the final processing step of the small ribosomal subunit RNA is the endonucleolytic cleavage of 20S pre-rRNA at cleavage site D yielding mature 18S rRNA. Depletion of Fap7 in yeast leads to a dramatic accumulation of 20S pre-rRNA and a concomitant decrease in 18S rRNA in the cytoplasm. In addition, these cells contain higher levels of 60S, but decreased numbers of 40S ribosomal subunits. Fap7 contains a P-loop like motif placing it in a class with NTPases and kinases and a role for it as an adenylate kinase has been suggested. Up to now both the structure of Fap7 and its detailed function during ribosome biogenesis remain elusive. Here, we present the backbone NMR assignments of a Fap7 homolog from the thermophilic archaeon Pyrococcus horikoshii in its nucleotide free form and bound to the adenylate kinase inhibitor AP5A.

Severe Acute Respiratory Syndrome Coronavirus Protein nsp1 Is a Novel Eukaryotic Translation Inhibitor That Represses Multiple Steps of Translation Initiation

Severe acute respiratory syndrome (SARS) coronavirus nonstructural protein 1 (nsp1) binds to the 40S ribosomal subunit and inhibits translation, and it also induces a template-dependent endonucleolytic cleavage of host mRNAs. nsp1 inhibits the translation of cap-dependent and internal ribosome entry site (IRES)-driven mRNAs, including SARS coronavirus mRNAs, hepatitis C virus (HCV), and cricket paralysis virus (CrPV) IRES-driven mRNAs that are resistant to nsp1-induced RNA cleavage. We used an nsp1 mutant, nsp1-CD, lacking the RNA cleavage function, to delineate the mechanism of nsp1-mediated translation inhibition and identify the translation step(s) targeted by nsp1. nsp1 and nsp1-CD had identical inhibitory effects on mRNA templates that are resistant to nsp1-induced RNA cleavage, implying the validity of using nsp1-CD to dissect the translation inhibition function of nsp1. We provide evidence for a novel mode of action of nsp1. nsp1 inhibited the translation initiation step by targeting at least two separate stages: 48S initiation complex formation and the steps involved in the formation of the 80S initiation complex from the 48S complex. nsp1 had a differential, mRNA template-dependent, inhibitory effect on 48S and 80S initiation complex formation. nsp1 inhibited different steps of translation initiation on CrPV and HCV IRES, both of which initiate translation via an IRES-40S binary complex intermediate; nsp1 inhibited binary complex formation on CrPV IRES and 48S complex formation on HCV IRES. Collectively, the data revealed that nsp1 inhibited translation by exerting its effect on multiple stages of translation initiation, depending on the mechanism of initiation operating on the mRNA template.

Differential Control of the Rate of 5′-End-Dependent mRNA Degradation in Escherichia coli

Many Escherichia coli mRNAs are degraded by a 5'-end-dependent mechanism in which RppH-catalyzed conversion of the 5'-terminal triphosphate to a monophosphate triggers rapid endonucleolytic cleavage by RNase E. However, little is understood about what governs the decay rates of these transcripts. We investigated the decay of three such messages--rpsT P1, yfcZ, and ydfG--to characterize the rate-determining step in their degradation. The steady-state ratio of monophosphorylated to triphosphorylated rpsT P1 and yfcZ mRNA indicates that their decay rate is limited by cleavage of the monophosphorylated intermediate, making RNase E critical for their rapid turnover. Conversely, the decay rate of ydfG is limited by generation of the monophosphorylated intermediate; therefore, either RNase E or its less abundant paralog RNase G is sufficient for rapid ydfG degradation. Although all three transcripts are stabilized when RppH is absent, overproducing RppH does not accelerate their decay, nor does RppH overproduction appear to influence the longevity of most other messages that it targets. The failure of excess RppH to hasten rpsT P1 and yfcZ degradation despite increasing the percentage of each that is monophosphorylated is consistent with the observation that pyrophosphate removal is not the rate-limiting step in their decay. In contrast, neither the ydfG decay rate nor the fraction of ydfG transcripts that are monophosphorylated increases when the cellular concentration of RppH is raised, suggesting that, for some RppH targets, the rate of formation of the monophosphorylated intermediate is limited by an ancillary factor or by a step that precedes pyrophosphate removal.

Transcriptome-Wide Characterization of miRNA-Directed and Non-miRNA-Directed Endonucleolytic Cleavage Using Degradome Analysis Under Low Ambient Temperature in Phalaenopsis aphrodite subsp. formosana

Plant microRNAs (miRNAs) regulate gene expression through post-transcriptional gene silencing. Phalaenopsis aphrodite subsp. formosana is an orchid species native to Taiwan, which has high economic value and a high frequency of floral polymorphism. To date, few studies have focused on the regulatory roles of miRNAs and functional small RNAs (sRNAs) in orchids although understanding the regulation of flower development and flowering time is potentially important. Here, we combined analyses of the transcriptome, sRNAs and the degradome to identify sRNA-directed transcript cleavages in Phalaenopsis. Degradome analysis provided large-scale evidence of conserved and novel miRNA-directed cleavage of target transcripts, and 46 abundant sRNA groups and their target transcripts were identified. Low temperature-responsive sRNAs were validated with normalized reads from an sRNA library and quantitative stem-loop reverse transcription-PCR (RT-PCR) analysis. According to gene ontology (GO) categorization, target transcripts of the novel miRNAs and sRNAs are functionally involved in metabolic processes or responses to stress. One particular homologous gene, Allcontig28452, which encodes digalactosyldiacylglycerol synthase 2 (DGD2), was found to be targeted by natural antisense transcripts (NATs) unique to Phalaenopsis. In summary, comprehensive analyses of the transcriptome, sRNAs and degradome using deep sequencing technology provided a useful platform for investigating miRNA-directed and non-miRNA-directed endonucleolytic cleavage in a non-model plant, the orchid Phalaenopsis.

Anticodon nuclease encoding virus-like elements in yeast

A variety of yeast species are known to host systems of cytoplasmic linear dsDNA molecules that establish replication and transcription independent of the nucleus via self-encoded enzymes that are phylogenetically related to those encoded by true infective viruses. Such yeast virus-like elements (VLE) fall into two categories: autonomous VLEs encode all the essential functions for their inheritance, and additional, dependent VLEs, which may encode a toxin-antitoxin system, generally referred to as killer toxin and immunity. In the two cases studied in depth, killer toxin action relies on chitin binding and hydrophobic domains, together allowing a separate toxic subunit to sneak into the target cell. Mechanistically, the latter sabotages codon-anticodon interaction by endonucleolytic cleavage of specific tRNAs 3' of the wobble nucleotide. This primary action provokes a number of downstream effects, including DNA damage accumulation, which contribute to the cell-killing efficiency and highlight the importance of proper transcript decoding capacity for other cellular processes than translation itself. Since wobble uridine modifications are crucial for efficient anticodon nuclease (ACNase) action of yeast killer toxins, the latter are valuable tools for the characterization of a surprisingly complex network regulating the addition of wobble base modifications in tRNA.

CSR-1 RNAi pathway positively regulates histone expression inC. elegans

Endogenous small interfering RNAs (endo-siRNAs) have been discovered in many organisms, including mammals. In C. elegans, depletion of germline-enriched endo-siRNAs found in complex with the CSR-1 Argonaute protein causes sterility and defects in chromosome segregation in early embryos. We discovered that knockdown of either csr-1, the RNA-dependent RNA polymerase (RdRP) ego-1, or the dicer-related helicase drh-3, leads to defects in histone mRNA processing, resulting in severe depletion of core histone proteins. The maturation of replication-dependent histone mRNAs, unlike that of other mRNAs, requires processing of their 3'UTRs through an endonucleolytic cleavage guided by the U7 snRNA, which is lacking in C. elegans. We found that CSR-1-bound antisense endo-siRNAs match histone mRNAs and mRNA precursors. Consistently, we demonstrate that CSR-1 directly binds to histone mRNA in an ego-1-dependent manner using biotinylated 2'-O-methyl RNA oligonucleotides. Moreover, we demonstrate that increasing the dosage of histone genes rescues the lethality associated with depletion of CSR-1 and EGO-1. These results support a positive and direct effect of RNAi on histone gene expression.

Potential role of tRNAs in wheat and Lolium mitochondrial rps7 transcript processing

The wheat mitochondrial gene for ribosomal protein S7 exhibits multiple transcripts that share the same 3' terminus but range in overall length from 3.4 to 0.7 kb because of 5'end-maturation events. The longest detectable precursor RNA maps precisely to the 3' end of a chloroplast-origin tRNA-Phe gene, consistent with it providing signals for endonucleolytic cleavage. Steady-state levels of precursor RNAs were seen to be lower in seedlings than in germinating embryos, although the degree of editing within untranslated regions (UTRs) was higher in seedlings. In another grass, Lolium multiflorum Lam., rps7 displays transcripts of 1.3 and 0.7 kb, and although the distal 5' UTRs are unrelated in sequence to those of wheat, the 5' terminus of the longer transcript also maps to a tRNA gene, in this case the native mitochondrial-type tRNA-Ser. Our findings illustrate the plasticity of plant mitochondrial transcriptional units and the recruitment of chloroplast-origin sequences for the expression of mitochondrial genes.

Severe Acute Respiratory Syndrome Coronavirus nsp1 Facilitates Efficient Propagation in Cells through a Specific Translational Shutoff of Host mRNA

Severe acute respiratory syndrome (SARS) coronavirus (SCoV) is an enveloped virus containing a single-stranded, positive-sense RNA genome. Nine mRNAs carrying a set of common 5' and 3' untranslated regions (UTR) are synthesized from the incoming viral genomic RNA in cells infected with SCoV. A nonstructural SCoV nsp1 protein causes a severe translational shutoff by binding to the 40S ribosomal subunits. The nsp1-40S ribosome complex further induces an endonucleolytic cleavage near the 5'UTR of host mRNA. However, the mechanism by which SCoV viral proteins are efficiently produced in infected cells in which host protein synthesis is impaired by nsp1 is unknown. In this study, we investigated the role of the viral UTRs in evasion of the nsp1-mediated shutoff. Luciferase activities were significantly suppressed in cells expressing nsp1 together with the mRNA carrying a luciferase gene, while nsp1 failed to suppress luciferase activities of the mRNA flanked by the 5'UTR of SCoV. An RNA-protein binding assay and RNA decay assay revealed that nsp1 bound to stem-loop 1 (SL1) in the 5'UTR of SCoV RNA and that the specific interaction with nsp1 stabilized the mRNA carrying SL1. Furthermore, experiments using an SCoV replicon system showed that the specific interaction enhanced the SCoV replication. The specific interaction of nsp1 with SL1 is an important strategy to facilitate efficient viral gene expression in infected cells, in which nsp1 suppresses host gene expression. Our data indicate a novel mechanism of viral gene expression control by nsp1 and give new insight into understanding the pathogenesis of SARS.

Global analysis of mRNA decay intermediates in Saccharomyces cerevisiae

The general pathways of eukaryotic mRNA decay occur via deadenylation followed by 3' to 5' degradation or decapping, although some endonuclease sites have been identified in metazoan mRNAs. To determine the role of endonucleases in mRNA degradation in Saccharomyces cerevisiae, we mapped 5' monophosphate ends on mRNAs in wild-type and dcp2 xrn1 yeast cells, wherein mRNA endonuclease cleavage products are stabilized. This led to three important observations. First, only few mRNAs that undergo low-level endonucleolytic cleavage were observed, suggesting that endonucleases are not a major contributor to yeast mRNA decay. Second, independent of known decapping enzymes, we observed low levels of 5' monophosphates on some mRNAs, suggesting that an unknown mechanism can generate 5' exposed ends, although for all substrates tested, Dcp2 was the primary decapping enzyme. Finally, we identified debranched lariat intermediates from intron-containing genes, demonstrating a significant discard pathway for mRNAs during the second step of pre-mRNA splicing, which is a potential step to regulate gene expression.

Molecular characterization of latent fertility restorer loci for Honglian cytoplasmic male sterility in Oryza species

In Oryza species, several distinct cytoplasmic male sterility (CMS) systems have been identified. Honglian (HL) is one of the mainstream breeding CMS lines in large-scale commercial production in China and southern Asia. In order to increase restorer diversity, we screened 130 Oryza accessions to find new restorer loci for HL-CMS. We analyzed the restoring ability and the diversity of molecular markers cosegregated with Rf5 (Milyang23) and Rf6(t) (9311) for HL-CMS. Molecular and genetic analyses revealed that eight accessions, including one cultivar and seven wild rice, showed polymerase chain reaction (PCR) patterns different from Rf5 and Rf6(t), and each of them harbored a pair of Rf genes unallelic to Rf5 and Rf6(t) for HL-CMS. Furthermore, Northern blot revealed that the Rf loci in the eight accessions showed three types of processing manners of HL-CMS-related transcripts, including endonucleolytic cleavage, partial endonucleolytic cleavage, and degradation of dicistronic atp6-orfH79 messenger RNA (mRNA). The Rf genes for HL-CMS in AA-genome Oryza species elucidated by genetic and molecular analyses will be highly beneficial to sustainable development of hybrid rice.

RNA degradation in Bacillus subtilis: an interplay of essential endo- and exoribonucleases.

RNA processing and degradation are key processes in the control of transcript accumulation and thus in the control of gene expression. In Escherichia coli, the underlying mechanisms and components of RNA decay are well characterized. By contrast, Gram-positive bacteria do not possess several important players of E. coli RNA degradation, most notably the essential enzyme RNase E. Recent research on the model Gram-positive organism, Bacillus subtilis, has identified the essential RNases J1 and Y as crucial enzymes in RNA degradation. While RNase J1 is the first bacterial exoribonuclease with 5'-to-3' processivity, RNase Y is the founding member of a novel class of endoribonucleases. Both RNase J1 and RNase Y have a broad impact on the stability of B. subtilis mRNAs; a depletion of either enzyme affects more than 25% of all mRNAs. RNases J1 and Y as well as RNase J2, the polynucleotide phosphorylase PNPase, the RNA helicase CshA and the glycolytic enzymes enolase and phosphofructokinase have been proposed to form a complex, the RNA degradosome of B. subtilis. This review presents a model, based on recent published data, of RNA degradation in B. subtilis. Degradation is initiated by RNase Y-dependent endonucleolytic cleavage, followed by processive exoribonucleolysis of the generated fragments both in 3'-to-5' and in 5'-to-3' directions. The implications of these findings for pathogenic Gram-positive bacteria are also discussed.

Non-coding RNA bioinformatics platform for full backing of the high-throughput sequencing experiments generated by next-generation sequencing technologies

Motivations. Short non-coding RNA molecules (20-30 nucleotides long) play an important role in the regulation of gene expression by interacting with their target RNAs. This interaction generally downregulates gene expression either affecting RNA stability or repressing translation. Different classes of small regulatory non coding RNAs (sncRNAs) have been discovered and studied so far, and new families continue to be described, which differ in the proteins required for their biogenesis, the mechanism of target recognition and regulation, and the biological pathways they control [1,2]. In particular, three major classes of sncRNAs have been mostly investigated: small interfering RNAs (siRNAs), micro-RNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) [1,2,3]. siRNAs direct the endonucleolytic cleavage of their target RNAs through a mechanism known as RNA interference (RNAi), miRNAs can repress translation or direct degradation of their target mRNA generally through imperfect complementary pairing on their 3'UTRs, whereas the major role of piRNAs is to ensure germline stability by repressing transposable elements (TEs). Recently, the advent of new Next-Generation Sequencing (NGS) technologies has awfully increased the throughput of transcriptome studies, thus allowing an unprecedented investigation of non-coding RNAs. Regulatory pathways involving ncRNAs, such as miRNAs, are now being elucidated in detail and functions for long non-coding RNAs are also emerging. The huge amount of transcript data produced by high-throughput sequencing requires the development and implementation of suitable bioinformatics workflows for their analysis and interpretation. Here we describe here a bioinformatics resource to classify and analyze the non-coding RNA component of human transcriptome sequence data obtained by different NGS platforms (Roche 454, Illumina and Solid). Methods. The ncRNAs bioinformatics platform is organized according to a typical three tier architecture: an analysis tier for ncRNAs detection, classification and functional analyses; a data tier made up of a data-warehouse used to store the analysis results, the ncRNAs reference database (a non-redundant collection of ncRNAs sequence retrieved from fRNAdb, RNAdb, mirBASE, NONCODE and others), the reference genome and other useful annotation database like HGNC nomenclature [4], Sequence Ontology (SO) [5] and Entrez Gene; a web tier module for querying the analysis results and the annotation stored in the ncRNAs reference database. The core of the platform is the analysis workflow. In figure 1 we show the pipeline for classification and functional annotations of non-coding RNAs (ncRNAs) fraction obtained through high-throughput sequencing (HTS) experiments using different NGS technologies. The input data for the bioinformatics platform can be either the reads data obtained by different NGS platforms (Roche 454, Illumina and Solid) or previously mapped reads stored in users' SAM/BAM files. Results. The ncRNA bioinformatics platform - through a combination of an analyses pipeline, a datawarehouse and a user-friendly web interface - is able to: i. detect and classify reads in known functional ncRNA categories using Sequence Ontology classification, HGNC nomenclature, gene names and miRNA accessions; ii. extract reads collections belonging to a givencategory for further analysis; iii. quantify ncRNA expression based on annotations derived from different reference ncRNA databases; iv. generate some statistics of expressed ncRNAs, indicating the RPKM (reads per kilobase of RNA model per million mapped reads)value for each Sequence Ontology class; v. detect differential expression of ncRNAs between two conditions (i.e. normal/pathological); vi. create a collections of interesting clusters of reads mapped on the genome but not detected as known ncRNA; vii. filter out reads mapping to ribosomal RNAs and mtDNA transcripts; viii. create a collection of unmapped residual reads (chimeras, artifacts, and contaminations).

Dom34:Hbs1 Plays a General Role in Quality-Control Systems by Dissociation of a Stalled Ribosome at the 3′ End of Aberrant mRNA

Translation arrest leads to an endonucleolytic cleavage of mRNA that is termed no-go decay (NGD). It has been reported that the Dom34:Hbs1 complex stimulates this endonucleolytic cleavage of mRNA induced by translation arrest in vivo and dissociates subunits of a stalled ribosome in vitro. Here we report that Dom34:Hbs1 dissociates the subunits of a ribosome that is stalled at the 3' end of mRNA in vivo, and has a crucial role in both NGD and nonstop decay. Dom34:Hbs1-mediated dissociation of a ribosome that is stalled at the 3' end of mRNA is required for degradation of a 5'-NGD intermediate. Dom34:Hbs1 facilitates the decay of nonstop mRNAs from the 3' end by exosomes and is required for the complete degradation of nonstop mRNA decay intermediates. We propose that Dom34:Hbs1 stimulates degradation of the 5'-NGD intermediate and of nonstop mRNA by dissociating the ribosome that is stalled at the 3' end of the mRNA.

The ubiquitous hammerhead ribozyme

The hammerhead ribozyme is a small catalytic RNA motif capable of endonucleolytic (self-) cleavage. It is composed of a catalytic core of conserved nucleotides flanked by three helices, two of which form essential tertiary interactions for fast self-scission under physiological conditions. Originally discovered in subviral plant pathogens, its presence in several eukaryotic genomes has been reported since. More recently, this catalytic RNA motif has been shown to reside in a large number of genomes. We review the different approaches in discovering these new hammerhead ribozyme sequences and discuss possible biological functions of the genomic motifs.