Introns within ribosomal protein genes regulate the production and function of yeast ribosomes.

In budding yeast, the most abundantly spliced pre-mRNAs encode ribosomal proteins (RPs). To investigate the contribution of splicing to ribosome production and function, we systematically eliminated introns from all RP genes to evaluate their impact on RNA expression, pre-rRNA processing, cell growth, and response to stress. The majority of introns were required for optimal cell fitness or growth under stress. Most introns are found in duplicated RP genes, and surprisingly, in the majority of cases, deleting the intron from one gene copy affected the expression of the other in a nonreciprocal manner. Consistently, 70% of all duplicated genes were asymmetrically expressed, and both introns and gene deletions displayed copy-specific phenotypic effects. Together, our results indicate that splicing in yeast RP genes mediates intergene regulation and implicate the expression ratio of duplicated RP genes in modulating ribosome function.

Reducing the structure bias of RNA-Seq reveals a large number of non-annotated non-coding RNA.

The study of RNA expression is the fastest growing area of genomic research. However, despite the dramatic increase in the number of sequenced transcriptomes, we still do not have accurate estimates of the number and expression levels of non-coding RNA genes. Non-coding transcripts are often overlooked due to incomplete genome annotation. In this study, we use annotation-independent detection of RNA reads generated using a reverse transcriptase with low structure bias to identify non-coding RNA. Transcripts between 20 and 500 nucleotides were filtered and crosschecked with non-coding RNA annotations revealing 111 non-annotated non-coding RNAs expressed in different cell lines and tissues. Inspecting the sequence and structural features of these transcripts indicated that 60% of these transcripts correspond to new snoRNA and tRNA-like genes. The identified genes exhibited features of their respective families in terms of structure, expression, conservation and response to depletion of interacting proteins. Together, our data reveal a new group of RNA that are difficult to detect using standard gene prediction and RNA sequencing techniques, suggesting that reliance on actual gene annotation and sequencing techniques distorts the perceived architecture of the human transcriptome.

snoDB: an interactive database of human snoRNA sequences, abundance and interactions.

Small nucleolar RNAs (snoRNAs) are an abundant type of non-coding RNA with conserved functions in all known eukaryotes. Classified into two main families, the box C/D and H/ACA snoRNAs, they enact their most well characterized role of guiding site specific modifications in ribosomal RNA, through the formation of specific ribonucleoprotein complexes, with fundamental implications in ribosome biogenesis. However, it is becoming increasingly clear that the landscape of snoRNA cellular functionality is much broader than it once seemed with novel members, non-uniform expression patterns, new and diverse targets as well as several emerging non-canonical functions ranging from the modulation of alternative splicing to the regulation of chromatin architecture. In order to facilitate the further characterization of human snoRNAs in a holistic manner, we introduce an online interactive database tool: snoDB. Its purpose is to consolidate information on human snoRNAs from different sources such as sequence databases, target information, both canonical and non-canonical from the literature and from high-throughput RNA-RNA interaction datasets, as well as high-throughput sequencing data that can be visualized interactively.

Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes.

Most budding yeast introns exist in the many duplicated ribosomal protein genes (RPGs) and it has been posited that they remain there to modulate the expression of RPGs and cell growth in response to stress. However, the mechanism by which introns regulate the expression of RPGs and their impact on the synthesis of ribosomal proteins remain unclear. In this study, we show that introns determine the ratio of ribosomal protein isoforms through asymmetric paralog-specific regulation of splicing. Exchanging the introns and 3' untranslated regions of the duplicated RPS9 genes altered the splicing efficiency and changed the ratio of the ribosomal protein isoforms. Mutational analysis of the RPS9 genes indicated that splicing is regulated by variations in the intron structure and the 3' untranslated region. Together these data suggest that preferential splicing of duplicated RPGs provides a means for adjusting the ratio of different ribosomal protein isoforms, while maintaining the overall expression level of each ribosomal protein.

Transcriptome wide annotation of eukaryotic RNase III reactivity and degradation signals.

Detection and validation of the RNA degradation signals controlling transcriptome stability are essential steps for understanding how cells regulate gene expression. Here we present complete genomic and biochemical annotations of the signals required for RNA degradation by the dsRNA specific ribonuclease III (Rnt1p) and examine its impact on transcriptome expression. Rnt1p cleavage signals are randomly distributed in the yeast genome, and encompass a wide variety of sequences, indicating that transcriptome stability is not determined by the recurrence of a fixed cleavage motif. Instead, RNA reactivity is defined by the sequence and structural context in which the cleavage sites are located. Reactive signals are often associated with transiently expressed genes, and their impact on RNA expression is linked to growth conditions. Together, the data suggest that Rnt1p reactivity is triggered by malleable RNA degradation signals that permit dynamic response to changes in growth conditions.

Yeast RNase III triggers polyadenylation-independent transcription termination.

Transcription termination of messenger RNA (mRNA) is normally achieved by polyadenylation followed by Rat1p-dependent 5'-3' exoribonuleolytic degradation of the downstream transcript. Here we show that the yeast ortholog of the dsRNA-specific ribonuclease III (Rnt1p) may trigger Rat1p-dependent termination of RNA transcripts that fail to terminate near polyadenylation signals. Rnt1p cleavage sites were found downstream of several genes, and the deletion of RNT1 resulted in transcription readthrough. Inactivation of Rat1p impaired Rnt1p-dependent termination and resulted in the accumulation of 3' end cleavage products. These results support a model for transcription termination in which cotranscriptional cleavage by Rnt1p provides access for exoribonucleases in the absence of polyadenylation signals.

Cancer-Associated Perturbations in Alternative Pre-messenger RNA Splicing.

For most of our 25,000 genes, the removal of introns by pre-messenger RNA (pre-mRNA) splicing represents an essential step toward the production of functional messenger RNAs (mRNAs). Alternative splicing of a single pre-mRNA results in the production of different mRNAs. Although complex organisms use alternative splicing to expand protein function and phenotypic diversity, patterns of alternative splicing are often altered in cancer cells. Alternative splicing contributes to tumorigenesis by producing splice isoforms that can stimulate cell proliferation and cell migration or induce resistance to apoptosis and anticancer agents. Cancer-specific changes in splicing profiles can occur through mutations that are affecting splice sites and splicing control elements, and also by alterations in the expression of proteins that control splicing decisions. Recent progress in global approaches that interrogate splicing diversity should help to obtain specific splicing signatures for cancer types. The development of innovative approaches for annotating and reprogramming splicing events will more fully establish the essential contribution of alternative splicing to the biology of cancer and will hopefully provide novel targets and anticancer strategies. Metazoan genes are usually made up of several exons interrupted by introns. The introns are removed from the pre-mRNA by RNA splicing. In conjunction with other maturation steps, such as capping and polyadenylation, the spliced mRNA is then transported to the cytoplasm to be translated into a functional protein. The basic mechanism of splicing requires accurate recognition of each extremity of each intron by the spliceosome. Introns are identified by the binding of U1 snRNP to the 5' splice site and the U2AF65/U2AF35 complex to the 3' splice site. Following these interactions, other proteins and snRNPs are recruited to generate the complete spliceosomal complex needed to excise the intron. While many introns are constitutively removed by the spliceosome, other splice junctions are not used systematically, generating the phenomenon of alternative splicing. Alternative splicing is therefore the process by which a single species of pre-mRNA can be matured to produce different mRNA molecules (Fig. 1). Depending on the number and types of alternative splicing events, a pre-mRNA can generate from two to several thousands different mRNAs leading to the production of a corresponding number of proteins. It is now believed that the expression of at least 70 % of human genes is subjected to alternative splicing, implying an enormous contribution to proteomic diversity, and by extension, to the development and the evolution of complex animals. Defects in splicing have been associated with human diseases (Caceres and Kornblihtt, Trends Genet 18(4):186-93, 2002, Cartegni et al., Nat Rev Genet 3(4):285-98, 2002, Pagani and Baralle, Nat Rev Genet 5(5):389-96, 2004), including cancer (Brinkman, Clin Biochem 37(7):584-94, 2004, Venables, Bioessays 28(4):378-86, 2006, Srebrow and Kornblihtt, J Cell Sci 119(Pt 13):2635-2641, 2006, Revil et al., Bull Cancer 93(9):909-919, 2006, Venables, Transworld Res Network, 2006, Pajares et al., Lancet Oncol 8(4):349-57, 2007, Skotheim and Nees, Int J Biochem Cell Biol 39:1432-1449, 2007). Numerous studies have now confirmed the existence of specific differences in the alternative splicing profiles between normal and cancer tissues. Although there are a few cases where specific mutations are the primary cause for these changes, global alterations in alternative splicing in cancer cells may be primarily derived from changes in the expression of RNA-binding proteins that control splice site selection. Overall, these cancer-specific differences in alternative splicing offer an immense potential to improve the diagnosis and the prognosis of cancer. This review will focus on the functional impact of cancer-associated alternative splicing variants, the molecular determinants that alter the splicing decisions in cancer cells, and future therapeutic strategies.

High-throughput quantification of splicing isoforms.

Most human messenger RNAs (mRNAs) are alternatively spliced and many exhibit disease-specific splicing patterns. However, the contribution of most splicing events to the development and maintenance of human diseases remains unclear. As the contribution of alternative splicing events to diagnosis and prognosis is becoming increasingly recognized, it becomes important to develop precise methods to quantify the abundance of these isoforms in clinical samples. Here we present a pipeline for real-time PCR annotation of splicing events (RASE) that allows accurate identification of a large number of splicing isoforms in human tissues. The RASE automatically designed specific primer pairs for 81% of all alternative splicing events in the NCBI build 36 database. Experimentally, the majority of the RASE designed primers resulted in isoform-specific amplification suitable for quantification in human cell lines or in formalin-fixed, paraffin-embedded (FFPE) RNA extract. Using this pipeline it is now possible to rapidly identify splicing isoform signatures in different types of human tissues or to validate complete sets of data generated by microarray expression profiling and deep sequencing techniques.

A G-tract element in apoptotic agents-induced alternative splicing.

Alternative splicing of a single pre-mRNA transcript can produce protein isoforms that promote either cell growth or death. Here we show that Ro-31-8220 (Ro), an apoptotic agent that inhibits protein kinase C and activates the c-Jun N terminal kinase, decreased the proportion of the cell growth-promoting Bcl-xL splice variant. Targeted mutagenesis analyses narrowed down a critical sequence to a 16-nt G-tract element (Gt16). Transferring this element to a heterologous gene conferred Ro response on an otherwise constitutive exon. The Ro effect was reduced by okadaic acid, an inhibitor of protein phosphatases PP1 and PP2A, in a concentration-dependent manner. Search in the human genome followed by RT-PCR identified a group of genes that contain similar exonic G-tract elements and are responsive to Ro. Moreover, the Gt16 element also mediates the regulation of alternative splicing by other cell apoptosis-inducers particularly retinoic acid. Therefore, the G-tract element likely plays a role in the apoptotic agents-induced alternative splicing of a group of genes. The functions of these genes imply that this regulation will have impact on cell growth/death.

Anticancer drugs affect the alternative splicing of Bcl-x and other human apoptotic genes.

Inducing an apoptotic response is the goal of most current chemotherapeutic interventions against cancer. However, little is known about the effect of chemotherapeutic agents on the alternative splicing of apoptotic genes. Here, we have tested 20 of the mainstream anticancer drugs for their ability to influence the production of Bcl-x splice isoforms. We find that many drugs shift splicing toward the proapoptotic Bcl-x(S) splice variant in 293 cells. The drugs modulate splicing decisions most likely through signaling events because the splicing switch is not compromised by inhibiting de novo protein synthesis or the activity of caspases. Several drugs also shift Bcl-x splicing in cancer cell lines (MCF-7, HeLa, PC-3, PA-1, and SKOV-3), but the set of active drugs varies between cell lines. We also examined the effect of anticancer agents on the alternative splicing of 95 other human apoptotic genes in different cell lines. Almost every drug can alter a subset of alternative splicing events in each cell line. Although drugs of the same class often influence the alternative splicing of the same units in individual cell lines, these units differ considerably between cell lines, indicating cell line-specific differences in the pathways that control splicing.

RNase III-mediated silencing of a glucose-dependent repressor in yeast.

Members of the RNase III family are found in all species examined with the exception of archaebacteria, where the functions of RNase III are carried out by the bulge-helix-bulge nuclease (BHB). In bacteria, RNase III contributes to the processing of many noncoding RNAs and directly cleaves several cellular and phage mRNAs. In eukaryotes, orthologs of RNase III participate in the biogenesis of many miRNAs and siRNAs, and this biogenesis initiates the degradation or translational repression of several mRNAs. However, the capacity of eukaryotic RNase IIIs to regulate gene expression by directly cleaving within the coding sequence of mRNAs remains speculative. Here we show that Rnt1p, a member of the RNase III family, selectively inhibits gene expression in baker's yeast by directly cleaving a stem-loop structure within the mRNA coding sequence. Analysis of mRNA expression upon the deletion of Rnt1p revealed an upregulation of the glucose-dependent repressor Mig2p. Mig2p mRNA became more stable upon the deletion of Rnt1p and resisted glucose-dependent degradation. In vitro, Rnt1p cleaved Mig2p mRNA and a silent mutation that disrupts Rnt1p signals blocked Mig2p mRNA degradation. These observations reveal a new RNase III-dependent mechanism of eukaryotic mRNA degradation.

Characterization of the reactivity determinants of a novel hairpin substrate of yeast RNase III.

RNase III enzymes form a conserved family of proteins that specifically cleave double-stranded (dsRNA). These proteins are involved in a variety of cellular functions, including the processing of many non-coding RNAs, mRNA decay, and RNA interference. Yeast RNase III (Rnt1p) selects its substrate by recognizing the structure generated by a conserved NGNN tetraloop (G2-loop). Mutations of the invariant guanosine stringently inhibit binding and cleavage of all known Rnt1p substrates. Surprisingly, we have found that the 5' end of small nucleolar RNA 48 is processed by Rnt1p in the absence of a G2-loop. Instead, biochemical and structural analyses revealed that cleavage, in this case, is directed by a hairpin capped with an AAGU tetraloop, with a preferred adenosine in the first position (A1-loop). Chemical probing indicated that A1-loops adopt a distinct structure that varies at the 3' end where Rnt1p interacts with G2-loops. Consistently, chemical footprinting and chemical interference assays indicate that Rnt1p binds to G2 and A1-loops using different sets of nucleotides. Also, cleavage and binding assays showed that the N-terminal domain of Rnt1p aids selection of A1-capped hairpins. Together, the results suggest that Rnt1p recognizes at least two distinct classes of tetraloops using flexible protein RNA interactions. This underscores the capacity of double-stranded RNA binding proteins to use several recognition motifs for substrate identification.

Structure of an AAGU tetraloop and its contribution to substrate selection by yeast RNase III.

RNase III enzymes are a highly conserved family of proteins that specifically cleave double-stranded RNA (dsRNA). These proteins are involved in a variety of cellular functions, including the processing of many non-coding RNAs, mRNA decay, and RNA interference. In yeast Rnt1p, a dsRNA-binding domain (dsRBD) recognizes its substrate by interacting with stems capped with conserved AGNN tetraloops. The enzyme uses the tetraloop to cut 14nt to 16nt away into the stem in a ruler-like mechanism. The solution structure of Rnt1p dsRBD complexed to one of its small nucleolar (sno) RNA substrate revealed non-sequence-specific contacts with the sugar-phosphate backbone in the minor groove of the AGNN fold and the two non-conserved tetraloop nucleotides. Recently, a new form of Rnt1p substrates lacking the conserved AGNN sequence but instead harboring an AAGU tetraloop was found at the 5' end of snoRNA 48 precursor. Here, we report the solution structure of this hairpin capped with an AAGU tetraloop. Some of the stacking interactions and the position of the turn in the sugar-phosphate backbone are similar to the one observed in the AGNN loop structure; however, the AAGU sequence adopts a different conformation. The most striking difference was found at the 3' end of the loop where Rnt1p interacts with AGNN substrates. The last nucleotide is extruded from the AAGU tetraloop structure in contrast to the compact AGNN fold. The AAGU hairpin structure suggests that Rnt1p recognizes substrates with different tetraloop structures, indicating that the structural repertoire specifically recognized by Rnt1p is larger than previously anticipated.

A physical interaction between Gar1p and Rnt1pi is required for the nuclear import of H/ACA small nucleolar RNA-associated proteins.

During rRNA biogenesis, multiple RNA and protein substrates are modified and assembled through the coordinated activity of many factors. In Saccharomyces cerevisiae, the double-stranded RNA nuclease Rnt1p and the H/ACA snoRNA pseudouridylase complex participate in the transformation of the nascent pre-rRNA transcript into 35S pre-rRNA. Here we demonstrate the binding of a component of the H/ACA complex (Gar1p) to Rnt1p in vivo and in vitro in the absence of other factors. In vitro, Rnt1p binding to Gar1p is mutually exclusive of its RNA binding and cleavage activities. Mutations in Rnt1p that disrupt Gar1p binding do not inhibit RNA cleavage in vitro but slow RNA processing, prevent nucleolar localization of H/ACA snoRNA-associated proteins, and reduce pre-rRNA pseudouridylation in vivo. These results demonstrate colocalization of various components of the rRNA maturation complex and suggest a mechanism that links rRNA pseudouridylation and cleavage factors.

Cell cycle-dependent nuclear localization of yeast RNase III is required for efficient cell division.

Members of the double-stranded RNA-specific ribonuclease III (RNase III) family were shown to affect cell division and chromosome segregation, presumably through an RNA interference-dependent mechanism. Here, we show that in Saccharomyces cerevisiae, where the RNA interference machinery is not conserved, an orthologue of RNase III (Rnt1p) is required for progression of the cell cycle and nuclear division. The deletion of Rnt1p delayed cells in both G1 and G2/M phases of the cell cycle. Nuclear division and positioning at the bud neck were also impaired in Deltarnt1 cells. The cell cycle defects were restored by the expression of catalytically inactive Rnt1p, indicating that RNA cleavage is not essential for cell cycle progression. Rnt1p was found to exit from the nucleolus to the nucleoplasm in the G2/M phase, and perturbation of its localization pattern delayed the progression of cell division. A single mutation in the Rnt1p N-terminal domain prevented its accumulation in the nucleoplasm and slowed exit from mitosis without any detectable effects on RNA processing. Together, the data reveal a new role for a class II RNase III in the cell cycle and suggest that at least some members of the RNase III family possess catalysis-independent functions.

RiboSubstrates: a web application addressing the cleavage specificities of ribozymes in designated genomes.

BACKGROUND: RNA-dependent gene silencing is becoming a routine tool used in laboratories worldwide. One of the important remaining hurdles in the selection of the target sequence, if not the most important one, is the designing of tools that have minimal off-target effects (i.e. cleaves only the desired sequence). Increasingly, in the current dawn of the post-genomic era, there is a heavy reliance on tools that are suitable for high-throughput functional genomics, consequently more and more bioinformatic software is becoming available. However, to date none have been designed to satisfy the ever-increasing need for the accurate selection of targets for a specific silencing reagent. RESULTS: In order to overcome this hurdle we have developed RiboSubstrates http://www.riboclub.org/ribosubstrates. This integrated bioinformatic software permits the searching of a cDNA database for all potential substrates for a given ribozyme. This includes the mRNAs that perfectly match the specific requirements of a given ribozyme, as well those including Wobble base pairs and mismatches. The results generated allow rapid selection of sequences suitable as targets for RNA degradation. The current web-based RiboSubstrates version permits the identification of potential gene targets for both SOFA-HDV ribozymes and for hammerhead ribozymes. Moreover, a minimal template for the search of siRNAs is also available. This flexible and reliable tool is easily adaptable for use with any RNA tool (i.e. other ribozymes, deoxyribozymes and antisense), and may use the information present in any cDNA bank. CONCLUSION: RiboSubstrates should become an essential step for all, even including "non-RNA biologists", who endeavor to develop a gene-inactivation system.

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals.

BACKGROUND: We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. RESULTS: We show that an oligonucleotide with a 5' tail containing the human beta-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. CONCLUSION: Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

A high-throughput method to measure the sensitivity of yeast cells to genotoxic agents in liquid cultures.

The sensitivity of yeast Saccharomyces cerevisiae to DNA damaging agents is better represented when cells are grown in liquid media than on solid plates. However, systematic assessment of several strains that are grown in different conditions is a cumbersome undertaking. We report an assay to determine cell growth based on automatic measurements of optical densities of very small (100 microl) liquid cell cultures. Furthermore, an algorithm was elaborated to analyze large data files obtained from the cell growth curves, which are described by the growth rate--that starts at zero and accelerates to the maximal rate (mu(m))--and by the lag time (lambda). Cell dilution spot test for colony formation on solid media and the growth curve assay were used in parallel to analyze the phenotypes of cells after treatments with three different classes of DNA damaging agents (methyl methanesulfonate, bleomycin, and ultraviolet light). In these experiments the survival of the WT (wild type) and a number of DNA repair-deficient strains were compared. The results show that only the cell growth curve assay could uncover subtle phenotypes when WT cells, or mutant strains that are only weakly affected in DNA repair proficiency, were treated with low doses of cytotoxic compounds. The growth curve assay was also applied to establish whether histone acetyltransferases and deacetylases affect the resistance of yeast cells to UV irradiation. Out of 20 strains tested the sir2delta and rpd3delta cells were found to be more resistant than the WT, while gcn5delta and spt10delta cells were found to be more sensitive. This new protocol is sensitive, provides quantifiable data, offers increased screening capability and speed compared to the colony formation test.

Free uptake of cell-penetrating peptides by fission yeast.

An increasing number of peptides translocate the plasma membrane of mammalian cells promising new avenues for drug delivery. However, only a few examples are known to penetrate the fungal cell wall. We compared the capacity of different fluorophore-labelled peptides to translocate into fission yeast and human cells and determined their intracellular distribution. Most of the 20 peptides tested were able to enter human cells, but only one, transportan 10 (TP10), efficiently penetrated fission yeast and was distributed uniformly inside the cells. The results show that the fungal cell wall may reduce, but does not block peptide uptake.

Sequence dependence of substrate recognition and cleavage by yeast RNase III.

Yeast Rnt1p is a member of the double-stranded RNA (dsRNA) specific RNase III family of endoribonucleases involved in RNA processing and RNA interference (RNAi). Unlike other RNase III enzymes, which recognize a variety of RNA duplexes, Rnt1p cleaves specifically RNA stems capped with the conserved AGNN tetraloop. This unusual substrate specificity challenges the established dogma for substrate selection by RNase III and questions the dsRNA contribution to recognition by Rnt1p. Here we show that the dsRNA sequence adjacent to the tetraloop regulates Rnt1p cleavage by interfering with RNA binding. In context, sequences surrounding the cleavage site directly influence the cleavage efficiency. Introduction of sequences that stabilize the RNA helix enhanced binding while reducing the turnover rate indicating that, unlike the tetraloop, Rnt1p binding to the dsRNA helix may become rate-limiting. These results suggest that Rnt1p activity is strictly regulated by a combination of primary and tertiary structural elements allowing a substrate-specific binding and cleavage efficiency.