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.

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.

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.

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.

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.

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.

Function and synthesis of small nucleolar RNAs.

Eukaryotic cells contain an extraordinarily complex population of small nucleolar RNAs (snoRNAs). During its brief lifetime, each human pre-rRNA molecule will transiently associate with approximately 150 different snoRNA species. In the past year our understanding of snoRNAs has been clarified by the recognition that the snoRNA population can be divided into a small number of groups which are structurally and functionally distinct. The two largest groups of snoRNAs direct the site-specific modification of the pre-rRNA at positions of 2'-O-methylation and pseudouridine formatio. Other groups of snoRNAs function in pre-rRNA cleavage and in the formation of the correct structure of the pre-rRNA.

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.

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.

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.

The U5 and U6 small nuclear RNAs as active site components of the spliceosome.

Five small nuclear RNAs (U1, U2, U4, U5, and U6) participate in precursor messenger RNA (pre-mRNA) splicing. To probe their interactions within the active center of the mammalian spliceosome, substrates containing a single photoactivatable 4-thiouridine residue adjacent to either splice site were synthesized, and crosslinks were induced during the course of in vitro splicing. An invariant loop sequence in U5 small nuclear RNA contacts exon 1 before and after the first step of splicing because a crosslink between U5 and the last residue of exon 1 appeared in the pre-mRNA and then in the cutoff exon 1 intermediate. Both of these crosslinked species could undergo subsequent splicing, indicating that the crosslinks reflect a functional interaction that is maintained through both reaction steps. The same U5 loop aligns the two exons for ligation since the first residue of exon 2 also became crosslinked to U5 in the lariat intermediate. An invariant sequence in U6 RNA became crosslinked to the conserved second position of the intron within both the lariat intermediate and the lariat intron product. On the basis of these results, several conformational arrangements of small nuclear RNAs within the spliceosomal active center can be distinguished, and additional mechanistic parallels between the spliceosome and self-splicing introns can be drawn.

Involvement of U6 snRNA in 5' splice site selection.

Two models describing the interaction between U6 small nuclear RNA (snRNA) and the 5' splice site of introns have been proposed on the basis of cross-linking experiments. Here it is shown that a conserved sequence present in U6 snRNA forms base pairs with conserved nucleotides at the 5' splice junction and that this interaction is involved in 5' splice site choice. These results demonstrate a specific function for U6 snRNA in splicing and suggest that U6 snRNA has a proofreading role during splice site selection. A model is presented in which this new interaction, in concert with previously described interactions between U6 snRNA, U2 snRNA, and the pre-messenger RNA, would position the branch point near the 5' splice site for the catalysis of the first splicing step.

Control of alternative splicing by the differential binding of U1 small nuclear ribonucleoprotein particle.

Cellular factors controlling alternative splicing of precursor messenger RNA are largely unknown, even though this process plays a central role in specifying the diversity of proteins in the eukaryotic cell. For the identification of such factors, a segment of the rat preprotachykinin gene was used in which differential expression of neuropeptides gamma and K is dependent on alternative splicing of the fourth exon (E4). Sequence variants of the three-exon segment, (E3-E4-E5) were created, resulting in a sensitive assay for factors mediating the splicing switch between E4-skipping and E4-inclusion. A dinucleotide mutation in the 5' splice site of E4 that increase base-pairing of this site to U1 small nuclear RNA resulted in uniform selection of E4, whereas a control mutation that destroyed base-pairing resulted in uniform E4-skipping. Affinity selection of spliceosomes formed on these functionally distinct substrates revealed that the extreme difference in splicing was mediated by differential binding of the U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site of E4. These data show that, apart from its established role in selecting 5' splice sites, U1 snRNP plays a fundamental role in 3' exon selection and provides insight into possible mechanisms of alternative splicing.

Requirement for intron-encoded U22 small nucleolar RNA in 18S ribosomal RNA maturation.

The nucleoli of vertebrate cells contain a number of small RNAs that are generated by the processing of intron fragments of protein-coding gene transcripts. The host gene (UHG) for intro-encoded human U22 is unusual in that it specifies a polyadenylated but apparently noncoding RNA. Depletion of U22 from Xenopus oocytes by oligonucleotide-directed ribonuclease H targeting prevented the processing of 18S ribosomal RNA (rRNA) at both ends. The appearance of 18S rRNA was restored by injection of in vitro-synthesized U22 RNA. These results identify a cellular function for an intron-encoded small RNA.

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.

The splicing factor Prp43p, a DEAH box ATPase, functions in ribosome biogenesis.

Biogenesis of the small and large ribosomal subunits requires modification, processing, and folding of pre-rRNA to yield mature rRNA. Here, we report that efficient biogenesis of both small- and large-subunit rRNAs requires the DEAH box ATPase Prp43p, a pre-mRNA splicing factor. By steady-state analysis, a cold-sensitive prp43 mutant accumulates 35S pre-rRNA and depletes 20S, 27S, and 7S pre-rRNAs, precursors to the small- and large-subunit rRNAs. By pulse-chase analysis, the prp43 mutant is defective in the formation of 20S and 27S pre-rRNAs and in the accumulation of 18S and 25S mature rRNAs. Wild-type Prp43p immunoprecipitates pre-rRNAs and mature rRNAs, indicating a direct role in ribosome biogenesis. The Prp43p-Q423N mutant immunoprecipitates 27SA2 pre-rRNA threefold more efficiently than the wild type, suggesting a critical role for Prp43p at the earliest stages of large-subunit biogenesis. Consistent with an early role for Prp43p in ribosome biogenesis, Prp43p immunoprecipitates the majority of snoRNAs; further, compared to the wild type, the prp43 mutant generally immunoprecipitates the snoRNAs more efficiently. In the prp43 mutant, the snoRNA snR64 fails to methylate residue C2337 in 27S pre-rRNA, suggesting a role in snoRNA function. We propose that Prp43p promotes recycling of snoRNAs and biogenesis factors during pre-rRNA processing, similar to its recycling role in pre-mRNA splicing. The dual function for Prp43p in the cell raises the possibility that ribosome biogenesis and pre-mRNA splicing may be coordinately regulated.