A single H/ACA small nucleolar RNA mediates tumor suppression downstream of oncogenic RAS.

Small nucleolar RNAs (snoRNAs) are a diverse group of non-coding RNAs that direct chemical modifications at specific residues on other RNA molecules, primarily on ribosomal RNA (rRNA). SnoRNAs are altered in several cancers; however, their role in cell homeostasis as well as in cellular transformation remains poorly explored. Here, we show that specific subsets of snoRNAs are differentially regulated during the earliest cellular response to oncogenic RASG12V expression. We describe a novel function for one H/ACA snoRNA, SNORA24, which guides two pseudouridine modifications within the small ribosomal subunit, in RAS-induced senescence in vivo. We find that in mouse models, loss of Snora24 cooperates with RASG12V to promote the development of liver cancer that closely resembles human steatohepatitic hepatocellular carcinoma (HCC). From a clinical perspective, we further show that human HCCs with low SNORA24 expression display increased lipid content and are associated with poor patient survival. We next asked whether ribosomes lacking SNORA24-guided pseudouridine modifications on 18S rRNA have alterations in their biophysical properties. Single-molecule Fluorescence Resonance Energy Transfer (FRET) analyses revealed that these ribosomes exhibit perturbations in aminoacyl-transfer RNA (aa-tRNA) selection and altered pre-translocation ribosome complex dynamics. Furthermore, we find that HCC cells lacking SNORA24-guided pseudouridine modifications have increased translational miscoding and stop codon readthrough frequencies. These findings highlight a role for specific snoRNAs in safeguarding against oncogenic insult and demonstrate a functional link between H/ACA snoRNAs regulated by RAS and the biophysical properties of ribosomes in cancer.

Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis.

Nuclear genes of euglenids and marine diplonemids harbor atypical, nonconventional introns which are not observed in the genomes of other eukaryotes. Nonconventional introns do not have the conserved borders characteristic for spliceosomal introns or the sequence complementary to U1 snRNA at the 5' end. They form a stable secondary structure bringing together both exon/intron junctions, nevertheless, this conformation does not resemble the form of self-splicing or tRNA introns. In the genes studied so far, frequent nonconventional introns insertions at new positions have been observed, whereas conventional introns have been either found at the conserved positions, or simply lost. In this work, we examined the order of intron removal from Euglena gracilis transcripts of the tubA and gapC genes, which contain two types of introns: nonconventional and spliceosomal. The relative order of intron excision was compared for pairs of introns belonging to different types. Furthermore, intermediate products of splicing were analyzed using the PacBio Next Generation Sequencing system. The analysis led to the main conclusion that nonconventional introns are removed in a rapid way but later than spliceosomal introns. Moreover, the observed accumulation of transcripts with conventional introns removed and nonconventional present may suggest the existence of a time gap between the two types of splicing.

Quantitative Activity Profile and Context Dependence of All Human 5' Splice Sites.

Pre-mRNA splicing is an essential step in the expression of most human genes. Mutations at the 5' splice site (5'ss) frequently cause defective splicing and disease due to interference with the initial recognition of the exon-intron boundary by U1 small nuclear ribonucleoprotein (snRNP), a component of the spliceosome. Here, we use a massively parallel splicing assay (MPSA) in human cells to quantify the activity of all 32,768 unique 5'ss sequences (NNN/GYNNNN) in three different gene contexts. Our results reveal that although splicing efficiency is mostly governed by the 5'ss sequence, there are substantial differences in this efficiency across gene contexts. Among other uses, these MPSA measurements facilitate the prediction of 5'ss sequence variants that are likely to cause aberrant splicing. This approach provides a framework to assess potential pathogenic variants in the human genome and streamline the development of splicing-corrective therapies.

Therapeutic activity of modified U1 core spliceosomal particles.

Modified U1 snRNAs bound to intronic sequences downstream of the 5' splice site correct exon skipping caused by different types of mutations. Here we evaluate the therapeutic activity and structural requirements of these exon-specific U1 snRNA (ExSpeU1) particles. In a severe spinal muscular atrophy, mouse model, ExSpeU1, introduced by germline transgenesis, increases SMN2 exon 7 inclusion, SMN protein production and extends life span. In vitro, RNA mutant analysis and silencing experiments show that while U1A protein is dispensable, the 70K and stem loop IV elements mediate most of the splicing rescue activity through improvement of exon and intron definition. Our findings indicate that precise engineering of the U1 core spliceosomal RNA particle has therapeutic potential in pathologies associated with exon-skipping mutations.

Analysis of aberrant pre-messenger RNA splicing resulting from mutations in ATP8B1 and efficient in vitro rescue by adapted U1 small nuclear RNA.

UNLABELLED: ATP8B1 deficiency is a severe autosomal recessive liver disease resulting from mutations in the ATP8B1 gene characterized by a continuous phenotypical spectrum from intermittent (benign recurrent intrahepatic cholestasis; BRIC) to progressive familial intrahepatic cholestasis (PFIC). Current therapeutic options are insufficient, and elucidating the molecular consequences of mutations could lead to personalized mutation-specific therapies. We investigated the effect on pre-messenger RNA splicing of 14 ATP8B1 mutations at exon-intron boundaries using an in vitro minigene system. Eleven mutations, mostly associated with a PFIC phenotype, resulted in aberrant splicing and a complete absence of correctly spliced product. In contrast, three mutations led to partially correct splicing and were associated with a BRIC phenotype. These findings indicate an inverse correlation between the level of correctly spliced product and disease severity. Expression of modified U1 small nuclear RNAs (snRNA) complementary to the splice donor sites strongly improved or completely rescued splicing for several ATP8B1 mutations located at donor, as well as acceptor, splice sites. In one case, we also evaluated exon-specific U1 snRNAs that, by targeting nonconserved intronic sequences, might reduce possible off-target events. Although very effective in correcting exon skipping, they also induced retention of the short downstream intron. CONCLUSION: We systematically characterized the molecular consequences of 14 ATP8B1 mutations at exon-intron boundaries associated with ATP8B1 deficiency and found that the majority resulted in total exon skipping. The amount of correctly spliced product inversely correlated with disease severity. Compensatory modified U1 snRNAs, complementary to mutated donor splice sites, were able to improve exon definition very efficiently and could be a novel therapeutic strategy in ATP8B1 deficiency as well as other genetic diseases.

Serotonin 2C receptor as a superhero: diversities and talents in the RNA universe for editing, variant, small RNA and other expected functional RNAs.

The serotonin 2C receptor subtype (5-HT2C) has a unique profession and continues to provide exciting and critical new information. The 5-HT2C is modulated at the RNA level by several mechanisms, including editing, short variant generation, and small RNAs. Recently, these phenomena, which had been demonstrated individually, were shown to be associated with each other. At present, many reports provide information about the influence of RNA regulation on receptor protein activities and expression, which was thought to be the final functional product. However, complicated behavior at the RNA stage allows us to imagine that the RNA itself has functional roles in the RNA universe. The 5-HT2C RNA may play several roles. This review will outline previous 5-HT2C studies and prospects for future studies.

An unanticipated early function of DEAD-box ATPase Prp28 during commitment to splicing is modulated by U5 snRNP protein Prp8.

The stepwise assembly of the highly dynamic spliceosome is guided by RNA-dependent ATPases of the DEAD-box family, whose regulation is poorly understood. In the canonical assembly model, the U4/U6.U5 triple snRNP binds only after joining of the U1 and, subsequently, U2 snRNPs to the intron-containing pre-mRNA. Catalytic activation requires the exchange of U6 for U1 snRNA at the 5' splice site, which is promoted by the DEAD-box protein Prp28. Because Prp8, an integral U5 snRNP protein, is thought to be a central regulator of DEAD-box proteins, we conducted a targeted search in Prp8 for cold-insensitive suppressors of a cold-sensitive Prp28 mutant, prp28-1. We identified a cluster of suppressor mutations in an N-terminal bromodomain-like sequence of Prp8. To identify the precise defect in prp28-1 strains that is suppressed by the Prp8 alleles, we analyzed spliceosome assembly in vivo and in vitro. Surprisingly, in the prp28-1 strain, we observed a block not only to spliceosome activation but also to one of the earliest steps of assembly, formation of the ATP-independent commitment complex 2 (CC2). The Prp8 suppressor partially corrected both the early assembly and later activation defects of prp28-1, supporting a role for this U5 snRNP protein in both the ATP-independent and ATP-dependent functions of Prp28. We conclude that the U5 snRNP has a role in the earliest events of assembly, prior to its stable incorporation into the spliceosome.

Minor introns are embedded molecular switches regulated by highly unstable U6atac snRNA.

Eukaryotes have two types of spliceosomes, comprised of either major (U1, U2, U4, U5, U6) or minor (U11, U12, U4atac, U6atac; <1%) snRNPs. The high conservation of minor introns, typically one amidst many major introns in several hundred genes, despite their poor splicing, has been a long-standing enigma. Here, we discovered that the low abundance minor spliceosome's catalytic snRNP, U6atac, is strikingly unstable (t½<2 hr). We show that U6atac level depends on both RNA polymerases II and III and can be rapidly increased by cell stress-activated kinase p38MAPK, which stabilizes it, enhancing mRNA expression of hundreds of minor intron-containing genes that are otherwise suppressed by limiting U6atac. Furthermore, p38MAPK-dependent U6atac modulation can control minor intron-containing tumor suppressor PTEN expression and cytokine production. We propose that minor introns are embedded molecular switches regulated by U6atac abundance, providing a novel post-transcriptional gene expression mechanism and a rationale for the minor spliceosome's evolutionary conservation. DOI:http://dx.doi.org/10.7554/eLife.00780.001.

The New Microbiology: a conference at the Institut de France.

In May 2012, three European Academies held a conference on the present and future of microbiology. The conference, entitled "The New Microbiology", was a joint effort of the French Académie des sciences, of the German National Academy of Sciences Leopoldina and of the British Royal Society. The organizers - Pascale Cossart and Philippe Sansonetti from the "Académie des sciences", David Holden and Richard Moxon from the "Royal Society", and Jörg Hacker and Jürgen Hesseman from the "Leopoldina Nationale Akademie der Wissenschaften" - wanted to highlight the current renaissance in the field of microbiology mostly due to the advent of technological developments and allowing for single-cell analysis, rapid and inexpensive genome-wide comparisons, sophisticated microscopy and quantitative large-scale studies of RNA regulation and proteomics. The conference took place in the historical Palais de l'Institut de France in Paris with the strong support of Jean-François Bach, Secrétaire Perpétuel of the Académie des sciences.

Selective silencing of mutated mRNAs in DM1 by using modified hU7-snRNAs.

We describe a function for modified human U7 small nuclear RNAs (hU7-snRNAs) distinct from modification of pre-mRNA splicing events. Engineered hU7-snRNAs containing a poly-CAG antisense sequence targeting the expanded CUG repeats of mutant DMPK transcripts in myotonic dystrophy caused specific degradation of pathogenic DMPK mRNAs without affecting the products of wild-type DMPK alleles. Abolition of the RNA gain-of-function toxicity that is responsible for pathogenesis supports the use of hU7-snRNAs for gene silencing in RNA-dominant disorders in which expanded repeats are expressed.

Role of the snRNAs in spliceosomal active site.

The spliceosome, the ribonucleoprotein assembly that removes the intervening sequences from pre-mRNAs through splicing, is one of the most complex cellular machines. In humans it is composed of -150 proteins and five RNAs (snRNAs). One of the snRNAs, U6, contains sequences analogous to all the RNA elements that form the active site of the group II introns, ribozymes that perform a splicing reaction mechanistically identical to spliceosomal splicing. Interestingly, U6 is the only snRNA that is indispensable for splicing and in vitro, in complex with another snRNA, it can catalyze a primordial splicing reaction in the absence of all other spliceosomal factors. On the other hand, discovery of an RNase H-like domain in a spliceosomal protein that is closely associated with splice sites suggests that proteins may be involved in formation of the active site. Thus, whether the spliceosome is an RNA or RNA-protein catalyst remains uncertain.

Post-transcriptional global regulation by CsrA in bacteria.

Global regulation allows bacteria to rapidly modulate the expression of a large variety of unrelated genes in response to environmental changes. Global regulators act at different levels of gene expression. This review focuses on CsrA, a post-transcriptional regulator that affects translation of its gene targets by binding mRNAs. CsrA controls a large variety of physiological processes such as central carbon metabolism, motility and biofilm formation. The activity of CsrA is itself tightly regulated by the CsrB and CsrC small RNAs and the BarA-UvrY two-component system.

Exhaustive analysis of the modular structure of the spliceosomal assembly network: a Petri net approach.

Spliceosomes are macro-complexes involving hundreds of proteins with many functional interactions. Spliceosome assembly belongs to the key processes that enable splicing of mRNA and modulate alternative splicing. A detailed list of factors involved in spliceosomal reactions has been assorted over the past decade, but, their functional interplay is often unknown and most of the present biological models cover only parts of the complete assembly process. It is a challenging task to build a computational model that integrates dispersed knowledge and combines a multitude of reaction schemes proposed earlier.Because for most reactions involved in spliceosome assembly kinetic parameters are not available, we propose a discrete modeling using Petri nets, through which we are enabled to get insights into the system's behavior via computation of structural and dynamic properties. In this paper, we compile and examine reactions from experimental reports that contribute to a functional spliceosome. All these reactions form a network, which describes the inventory and conditions necessary to perform the splicing process. The analysis is mainly based on system invariants. Transition invariants (T-invariants) can be interpreted as signaling routes through the network. Due to the huge number of T-invariants that arise with increasing network size and complexity, maximal common transition sets (MCTS) and T-clusters were used for further analysis. Additionally, we introduce a false color map representation, which allows a quick survey of network modules and the visual detection of single reactions or reaction sequences, which participate in more than one signaling route. We designed a structured model of spliceosome assembly, which combines the demands on a platform that i) can display involved factors and concurrent processes, ii) offers the possibility to run computational methods for knowledge extraction, and iii) is successively extendable as new insights into spliceosome function are reported by experimental reports. The network consists of 161 transitions (reactions) and 140 places (reactants). All reactions are part of at least one of the 71 T-invariants. These T-invariants define pathways, which are in good agreement with the current knowledge and known hypotheses on reaction sequences during spliceosome assembly, hence contributing to a functional spliceosome. We demonstrate that present knowledge, in particular of the initial part of the assembly process, describes parallelism and interaction of signaling routes, which indicate functional redundancy and reflect the dependency of spliceosome assembly initiation on different cellular conditions. The complexity of the network is further increased by two switches, which introduce alternative routes during A-complex formation in early spliceosome assembly and upon transition from the B-complex to the C-complex. By compiling known reactions into a complete network, the combinatorial nature of invariant computation leads to pathways that have previously not been described as connected routes, although their constituents were known. T-clusters divide the network into modules, which we interpret as building blocks in spliceosome maturation. We conclude that Petri net representations of large biological networks and system invariants, are well-suited as a means for validating the integration of experimental knowledge into a consistent model. Based on this network model, the design of further experiments is facilitated.

The C53/C37 subcomplex of RNA polymerase III lies near the active site and participates in promoter opening.

The C53 and C37 subunits of RNA polymerase III (pol III) form a subassembly that is required for efficient termination; pol III lacking this subcomplex displays increased processivity of RNA chain elongation. We show that the C53/C37 subcomplex additionally plays a role in formation of the initiation-ready open promoter complex similar to that of the Brf1 N-terminal zinc ribbon domain. In the absence of C53 and C37, the transcription bubble fails to stably propagate to and beyond the transcriptional start site even when the DNA template is supercoiled. The C53/C37 subcomplex also stimulates the formation of an artificially assembled elongation complex from its component DNA and RNA strands. Protein-RNA and protein-DNA photochemical cross-linking analysis places a segment of C53 close to the RNA 3' end and transcribed DNA strand at the catalytic center of the pol III elongation complex. We discuss the implications of these findings for the mechanism of transcriptional termination by pol III and propose a structural as well as functional correspondence between the C53/C37 subcomplex and the RNA polymerase II initiation factor TFIIF.

Rescue of coagulation factor VII function by the U1+5A snRNA.

Our previous studies with genomic minigenes have demonstrated that an engineered small nuclear RNA-U1 (U1+5a) partially rescued coagulation factor VII (FVII) mRNA processing impaired by the 9726+5G>A mutation. Here, to evaluate the U1+5a effects on FVII function, we devised a full-length FVII splicing-competent construct (pSCFVII-wt). This construct drove in COS-1 cells the synthesis of properly processed FVII transcripts and of secreted functional FVII (23 +/- 4 ng/mL), which were virtually undetectable upon introduction of the 9726+5G>A mutation (pSCFVII-9726+5a). Cotransfection of pSCFVII-9726+5a with pU1+5a resulted in a partial rescue of FVII splicing and protein biosynthesis. The level increase in medium was dose dependent and, with a molar excess (1.5x) of pU1+5a, reached 9.5% plus or minus 3.2% (5.0 +/- 2.8 ng/mL) of FVII-wt coagulant activity. These data provide the first insights into the U1-snRNA-mediated rescue of donor splice sites at protein level, thus further highlighting its therapeutic implications in bleeding disorders, which would benefit even from tiny increase of functional levels.

An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles.

NEAT1 RNA, a highly abundant 4 kb ncRNA, is retained in nuclei in approximately 10 to 20 large foci that we show are completely coincident with paraspeckles, nuclear domains implicated in mRNA nuclear retention. Depletion of NEAT1 RNA via RNAi eradicates paraspeckles, suggesting that it controls sequestration of the paraspeckle proteins PSP1 and p54, factors linked to A-I editing. Unlike overexpression of PSP1, NEAT1 overexpression increases paraspeckle number, and paraspeckles emanate exclusively from the NEAT1 transcription site. The PSP-1 RNA binding domain is required for its colocalization with NEAT1 RNA in paraspeckles, and biochemical analyses support that NEAT1 RNA binds with paraspeckle proteins. Unlike other nuclear-retained RNAs, NEAT1 RNA is not A-I edited, consistent with a structural role in paraspeckles. Collectively, results demonstrate that NEAT1 functions as an essential structural determinant of paraspeckles, providing a precedent for a ncRNA as the foundation of a nuclear domain.

Biochemical and biophysical analyses of concerted (U5/U3) integration.

Retrovirus integrase (IN) integrates the viral linear DNA genome ( approximately 10 kb) into a host chromosome, a step which is essential for viral replication. Integration occurs via a nucleoprotein complex, termed the preintegration complex (PIC). This article focuses on the reconstitution of synaptic complexes from purified components whose molecular properties mirror those of the PIC, including the efficient concerted integration of two ends of linear viral DNA into target DNA. The methods described herein permit the biochemical and biophysical analyses of concerted integration. The methods enable (1) the study of interactions between purified recombinant IN and its viral DNA substrates at the molecular level; (2) the identification and characterization of nucleoprotein complexes involved in the human immunodeficiency virus type-1 (HIV-1) concerted integration pathway; (3) the determination of the multimeric state of IN within these complexes; (4) dissection of the interaction between HIV-1 IN and cellular proteins such as lens epithelium-derived growth factor (LEDGF/p75); (5) the examination of HIV-1 Class II and strand transfer inhibitor resistant IN mutants; (6) the mechanisms associated with strand transfer inhibitors directed against HIV-1 IN that have clinical relevance in the treatment of HIV-1/AIDS.

The RNA continent.

Recent progress in the analyses of the mouse transcriptome leads to unexpected discoveries. The mouse genomic sequences read by RNA polymerase II may be six times more than previously expected for human chromosomes. The transcript-abundant regions (named "transcription forests") occupy more than half of the genomic sequence and are divided by transcript-scarce regions (transcription deserts). Many of the coding mRNAs may have partially overlapping antisense RNAs. There are transcripts bridging several adjacent genes that were previously regarded as distinct ones. The transcription start sites appearing as cap analysis of gene expression (CAGE) tags are mapped on the mouse genomic sequences. Distributions of CAGE tags show that the shapes of mammalian gene promoters can be classified into four major categories. These shapes were conserved between mouse and human. Most of the gene has exonic transcription start sites, especially in the 3' untranslated region (3' UTR) sequences. The term "RNA continent" has been invented to express this unexpectedly complex and prodigious mouse transcriptome. More than a half of the RNA polymerase II transcripts are regarded as noncoding RNAs (ncRNAs). The great variety of ncRNAs in mammalian transcriptome implies that there are many functional ncRNAs in the cells. Especially, the evolutionarily conserved microRNAs play critical roles in mammalian development and other biological functions. Moreover, many other ncRNAs have also been shown to have biological significant functions, mainly in the regulation of gene expression. The functional survey of the RNA continent has just started. We will describe the state of the art of the RNA continent and its impact on the modern molecular biology, especially on the cancer research.

MicroRNAs play an essential role in the development of cardiac hypertrophy.

MicroRNAs are naturally existing, small, noncoding RNA molecules that downregulate posttranscriptional gene expression. Their expression pattern and function in the heart remain unknown. Here we report an array of microRNAs that are differentially and temporally regulated during cardiac hypertrophy. Significantly, the muscle-specific microRNA-1 (miR-1) was singularly downregulated as early as day 1 (0.56+/-0.036), persisting through day 7 (0.29+/-0.14), after aortic constriction-induced hypertrophy in a mouse model. Overexpression experiments showed that miR-1 inhibited its in silico-predicted, growth-related targets, including Ras GTPase-activating protein (RasGAP), cyclin-dependent kinase 9 (Cdk9), fibronectin, and Ras homolog enriched in brain (Rheb), in addition to protein synthesis and cell size. Thus, we propose that microRNAs play an essential regulatory role in the development of cardiac hypertrophy, wherein downregulation of miR-1 is necessary for the relief of growth-related target genes from its repressive influence and induction of hypertrophy.