Ribonucleoprotein Particles Research Articles

Global Promotion of Alternative Internal Exon Usage by mRNA 3′ End Formation Factors

The mechanisms that regulate alternative precursor mRNA (pre-mRNA) splicing are largely unknown. Here, we perform an RNAi screen to identify factors required for alternative splicing regulation by RBFOX2, an RNA-binding protein that promotes either exon inclusion or exclusion. Unexpectedly, we find that two mRNA 3' end formation factors, cleavage and polyadenylation specificity factor (CPSF) and SYMPK, are RBFOX2 cofactors for both inclusion and exclusion of internal exons. RBFOX2 interacts with CPSF/SYMPK and recruits it to the pre-mRNA. RBFOX2 and CPSF/SYMPK then function together to regulate binding of the early intron recognition factors U2AF and U1 small nuclear ribonucleoprotein particle (snRNP). Genome-wide analysis reveals that CPSF also mediates alternative splicing of many internal exons in the absence of RBFOX2. Accordingly, we show that CPSF/SYMPK is also a cofactor of NOVA2 and heterologous nuclear ribonucleoprotein A1 (HNRNPA1), RNA-binding proteins that also regulate alternative splicing. Collectively, our results reveal an unanticipated role for mRNA 3' end formation factors in global promotion of alternative splicing.

RNA size is a critical factor for U-containing substrate selectivity and permanent pseudouridylated product release during the RNA:Ψ-synthase reaction catalyzed by box H/ACA sRNP enzyme at high temperature.

The box H/ACA small ribonucleoprotein particles (H/ACA sRNPs) are RNP enzymes that isomerize uridines (U) into pseudouridines (Ψ) in archaeal RNAs. The RNA component acts as a guide by forming base-pair interactions with the substrate RNA to specify the target nucleotide of the modification to the catalytic subunit Cbf5. Here, we have analyzed association of an H/ACA sRNP enzyme from the hyperthermophilic archaeon Pyrococcus abyssi with synthetic substrate RNAs of different length and with target nucleotide variants, and estimated their turnover at high temperature. In these conditions, we found that a short substrate, which length is restricted to the interaction with RNA guide sequence, has higher turnover rate. However, the longer substrate with additional 5' and 3' sequences non-complementary to the guide RNA is better discriminated by the U to Ψ conversion allowing the RNP enzyme to distinguish the modified product from the substrate. In addition, we identified that the conserved residue Y179 in the catalytic center of Cbf5 is crucial for substrate selectivity.

Independent and coordinate trafficking of single Drosophila germ plasm mRNAs.

mRNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. While different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single molecule imaging, we analyzed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells. We find that the germ cell-destined transcripts nanos, cyclin B, and polar granule component travel within the oocyte as ribonucleoprotein particles containing single mRNA molecules but co-assemble into multi-copy heterogeneous granules selectively at the posterior of the oocyte. The stoichiometry and dynamics of assembly indicate a defined stepwise sequence. Our data suggest that co-packaging of these transcripts ensures their effective segregation to germ cells. In contrast, compartmentalization of the germline determinant oskar mRNA into different granules limits its entry into germ cells. This exclusion is required for proper germline development.

Characterization of the Small Splicing Protein Dib1, a Component of the U4/U6‐U5 snRNP

The spliceosome is a highly dynamic molecular machine composed of small nuclear ribonucleoprotein particles (snRNP). The snRNP facilitates the joining of exons via two transesterification reactions that remove the non‐coding intronic regions. Once the introns are removed, the resulting mature RNA (mRNA) transcript can then be translated into functional protein. The fidelity of this process is crucial, and mistakes in splicing can lead to diseases such as Retinitis pigmentosa. One of the critical steps in splicing is the activation of the U4/U6‐U5 triple snRNP upon joining the spliceosomal complex.­­ In this study, we focus on the small, essential splicing protein Dib1, whose function in splicing remains unknown. Dib1 is 15kDa protein with a thioredoxin‐like fold. Previous data suggests that Dib1 interacts with splicing proteins that bridge the U4/U6 and U5 snRNPs. We characterized the role of Dib1 by rationally designing dib1 mutants and assaying for effects on Saccharomyces cerevisiae growth. We purified these Dib1 mutant proteins and examined their structures by circular dichroism in order to determine the effects these mutants have on the structure of Dib1 protein. In addition, splicing assays were conducted in order to determine the effects the mutant may have on mediating splicing. These assays will help further characterize Dib1 and its role in splicing as well as lend insight into further understanding the splicing machinery.

Nuclear export of messenger RNA.

Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex.

Defective structural RNA processing in relapsing-remitting multiple sclerosis.

BackgroundSurveillance of integrity of the basic elements of the cell including DNA, RNA, and proteins is a critical element of cellular physiology. Mechanisms of surveillance of DNA and protein integrity are well understood. Surveillance of structural RNAs making up the vast majority of RNA in a cell is less well understood. Here, we sought to explore integrity of processing of structural RNAs in relapsing remitting multiple sclerosis (RRMS) and other inflammatory diseases.ResultsWe employed mononuclear cells obtained from subjects with RRMS and cell lines. We used quantitative-PCR and whole genome RNA sequencing to define defects in structural RNA surveillance and siRNAs to deplete target proteins. We report profound defects in surveillance of structural RNAs in RRMS exemplified by elevated levels of poly(A) + Y1-RNA, poly(A) + 18S rRNA and 28S rRNAs, elevated levels of misprocessed 18S and 28S rRNAs and levels of the U-class of small nuclear RNAs. Multiple sclerosis is also associated with genome-wide defects in mRNA splicing. Ro60 and La proteins, which exist in ribonucleoprotein particles and play different roles in quality control of structural RNAs, are also deficient in RRMS. In cell lines, silencing of the genes encoding Ro60 and La proteins gives rise to these same defects in surveillance of structural RNAs.ConclusionsOur results establish that profound defects in structural RNA surveillance exist in RRMS and establish a causal link between Ro60 and La proteins and integrity of structural RNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0629-x) contains supplementary material, which is available to authorized users.

Co‐transcriptional mRNP formation is coordinated within a molecular mRNP packaging station in S. cerevisiae

In eukaryotes, the messenger RNA (mRNA), the blueprint of a protein‐coding gene, is processed and packaged into a messenger ribonucleoprotein particle (mRNP) by mRNA‐binding proteins in the nucleus. The steps of mRNP formation – transcription, processing, packaging, and the orchestrated release of the export‐competent mRNP from the site of transcription for nuclear mRNA export – are tightly coupled to ensure a highly efficient and regulated process. The importance of highly accurate nuclear mRNP formation is illustrated by the fact that mutations in components of this pathway lead to cellular inviability or to severe diseases in metazoans. We hypothesize that efficient mRNP formation is realized by a molecular mRNP packaging station, which is built by several recruitment platforms and coordinates the individual steps of mRNP formation.

RNPs and autoimmunity: 20 years later.

RNPs and autoimmunity: 20 years later.

Twenty years of RNA: reflections from the RNP world.

Twenty years of RNA: reflections from the RNP world.

In the beginning was the U1A protein: a personal reflection.

In the beginning was the U1A protein: a personal reflection.

Study of mRNA turnover never decays.

Study of mRNA turnover never decays.

Mechanisms and Regulation of Alternative Pre-mRNA Splicing.

Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA-protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA-RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions.

A role for vault particles as mediator for therapeutic effects against endocrine tumors

[08:40 – 08:50]The vault complex, consisting of a major vault protein (MVP), two minor vault proteins (VPARP and TEP1) and small untranslated RNA molecules (vault RNAs 1 – 4), is considered the largest intracellular ribonucleoprotein particle. Although in recent years, vaults were believed to be involved in multidrug resistance (MDR), the exact function of this complex has remained unclear. Recently, we investigated the therapeutic applicability of a Tumor-Vascular-Disrupting Agent (ASA) in preclinical models for neuroendocrine tumors of the gastroenteropancreatic system (BON) and adrenocortical carcinoma (NCIh295R). Upon treatment we detected highly significant anti-tumoral effects in BON xenografts while no effects were detectable for NCI-H295R. In an attempt to explain differences in therapeutic responsiveness, gene expression patterns within these tumors was investigated by an affymetrix gene array. Subsequent analyses identified vault RNAs 1 – 3 as the most pronounced regulated transcripts. Interestingly, we detected increased vault RNA expression in the tumor model showing therapeutic responsiveness. Quantitative Real Time PCR confirmed these results for ASA treated BON tumors (% of 100% controls; vault1: 2468.1 ± 367%, p < 0.001; vault2: 1922.5 ± 235%, p < 0.001; vault3: 901.3 ± 119%, p < 0.001) while no changes were detectable for the not-responding NCI-H295R-xenografts (% of 100% controls; vault1: 224.1 ± 84%, p > 0.05; vault2: 113 ± 23% p > 0.05; vault3: 89 ± 14% p > 0.05). Subsequently, we investigated NCI-H295R-xenografts which had been treated with two different chemotherapeutic regimens (EDP-M and LEDP-M). In this therapeutic setting treatment-dependent upregulation of vault RNAs could also be detected in NCI-H295R-tumors (% of 100% controls; vault1: EDP-M 296.3 ± 44%, p < 0.001; LEDP-M 243.5 ± 16%, p < 0.001; vault2: EDP-M 157.2 ± 20%, p > 0.05; LEDP-M 160.7 ± 20%, p > 0.05; vault3: EDP-M 241.2 ± 55%, p < 0.001; LEDP-M 118.9 ± 16%, p > 0.05). Further in vitro analyses revealed dynamic changes in vault RNA expression also upon blasticidin, doxorubicin, mitotane treatment and a significant increase in MVP protein levels upon TNF alpha treatment in BON cells. In summary, further investigation and manipulation of vault particles might have potential to improve efficacy of anticancer drugs in endocrine tumors.

Dramatically reduced spliceosome in Cyanidioschyzon merolae

The human spliceosome is a large ribonucleoprotein complex that catalyzes pre-mRNA splicing. It consists of five snRNAs and more than 200 proteins. Because of this complexity, much work has focused on the Saccharomyces cerevisiae spliceosome, viewed as a highly simplified system with fewer than half as many splicing factors as humans. Nevertheless, it has been difficult to ascribe a mechanistic function to individual splicing factors or even to discern which are critical for catalyzing the splicing reaction. We have identified and characterized the splicing machinery from the red alga Cyanidioschyzon merolae, which has been reported to harbor only 26 intron-containing genes. The U2, U4, U5, and U6 snRNAs contain expected conserved sequences and have the ability to adopt secondary structures and form intermolecular base-pairing interactions, as in other organisms. C. merolae has a highly reduced set of 43 identifiable core splicing proteins, compared with ∼90 in budding yeast and ∼140 in humans. Strikingly, we have been unable to find a U1 snRNA candidate or any predicted U1-associated proteins, suggesting that splicing in C. merolae may occur without the U1 small nuclear ribonucleoprotein particle. In addition, based on mapping the identified proteins onto the known splicing cycle, we propose that there is far less compositional variability during splicing in C. merolae than in other organisms. The observed reduction in splicing factors is consistent with the elimination of spliceosomal components that play a peripheral or modulatory role in splicing, presumably retaining those with a more central role in organization and catalysis.

Perturbation of ribosome biogenesis drives cells into senescence through 5S RNP-mediated p53 activation.

The 5S ribonucleoprotein particle (RNP) complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses.

An intimate view of a spliceosome component.

A high-resolution structure reveals how the ribonucleoprotein particle called U1 snRNP engages with 5′ splice sites.

Differences in the ovine HSP90AA1 gene expression rates caused by two linked polymorphisms at its promoter affect rams sperm DNA fragmentation under environmental heat stress conditions.

Heat shock (HS) is one of the best-studied exogenous cellular stresses. Almost all tissues, cell types, metabolic pathways and biochemical reactions are affected in greater or lesser extent by HS. However, there are some especially thermo sensible cellular types such as the mammalian male germ cells. The present study examined the role of three INDELs in conjunction with the -660G/C polymorphism located at the HSP90AA1 promoter region over the gene expression rate under HS. Specially, the -668insC INDEL, which is very close to the -660G/C transversion, is a good candidate to be implied in the transcriptional regulation of the gene by itself or in a cooperative way with this SNP. Animals carrying the genotype II-668 showed higher transcription rates than those with ID-668 (FC = 3.07) and DD-668 (FC = 3.40) genotypes for samples collected under HS. A linkage between gene expression and sperm DNA fragmentation was also found. When HS conditions were present along or in some stages of the spermatogenesis, alternative genotypes of the -668insC and -660G/C mutations are involved in the effect of HS over sperm DNA fragmentation. Thus, unfavorable genotypes in terms of gene expression induction (ID-668GC-660 and DD-668GG-660) do not produce enough mRNA (stored as messenger ribonucleoprotein particles) and Hsp90α protein to cope with future thermal stress which might occur in posterior stages when transcriptional activity is reduced and cell types and molecular processes are more sensible to heat (spermatocytes in pachytene and spermatids protamination). This would result in the impairment of DNA packaging and the consequent commitment of the events occurring shortly after fertilization and during embryonic development. In the short-term, the assessment of the relationship between sperm DNA fragmentation sensitivity and ram’s fertility will be of interest to a better understanding of the mechanisms of response to HS and its consequences on animal production and reproduction performance.

Paradigms of ribosome synthesis: Lessons learned from ribosomal proteins.

The proteome in all cells is manufactured via the intricate process of translation by multimolecular factories called ribosomes. Nevertheless, these ribonucleoprotein particles, the largest of their kind, also have an elaborate assembly line of their own. Groundbreaking discoveries that bacterial ribosomal subunits can be self-assembled in vitro jumpstarted studies on how ribosomes are constructed. Until recently, ribosome assembly has been investigated almost entirely in vitro with bacterial small subunits under equilibrium conditions. In light of high-resolution ribosome structures and a more sophisticated toolkit, the past decade has been defined by a burst of kinetic studies in vitro and, importantly, also a shift to examining ribosome maturation in living cells, especially in eukaryotes. In this review, we summarize the principles governing ribosome assembly that emerged from studies focusing on ribosomal proteins and their interactions with rRNA. Understanding these paradigms has taken center stage, given the linkage between anomalous ribosome biogenesis and proliferative disorders.

Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition.

U1 snRNP binds to the 5' exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing. We present two crystal structures of engineered U1 sub-structures, which together reveal at atomic resolution an almost complete network of protein-protein and RNA-protein interactions within U1 snRNP, and show how the 5' splice site of pre-mRNA is recognised by U1 snRNP. The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5'-end of U1 snRNA. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA. The structure, together with RNA binding assays, shows that the selection of 5'-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5'-splice sites.

Viral Membrane Fusion at Single Pore Resolution

Fusion between membrane-enveloped viruses and their hosts is a critical step in infection, enabling the virus to release its genome and subsequently hijack the host cell's replication machinery. The genomes of enveloped viruses are packaged into multiple bulky ribonucleoprotein (RNP) particles whose release requires the dilation of the fusion pore - the initial, narrow, connection between fusing membranes. Electrophysiological studies of influenza A hemagglutinin (HA)-mediated fusion between HA-expressing fibroblasts and red blood cells and between HA-fibroblasts and voltage-clamped suspended bilayers comprise the most complete information to date about the dynamics of viral fusion pores. Small pores (∼2-5nm) were found to flicker repetitively and/or fluctuate in size prior to terminal dilation. A quantitative understanding of the biophysical driving forces of viral membrane fusion, including membrane contact area, membrane tension and protein fusogen cooperativity, however, remains elusive, and a number of questions - regarding the physical basis of pore flickering and the number of HA trimers required for successful pore dilation - remain open. Here we describe a novel electrophysiological approach, which has been successfully applied to study SNARE-mediated fusion pores, to probe the HA-mediated fusion pore. We voltage-clamped HA-expressing fibroblasts in the cell-attached configuration and included nanodiscs (NDs) in the patch pipette as fusion partners to probe fusion pore nucleation and evolution at single pore resolution. NDs are flat, bilayer discs whose composition and size can be varied. Current through voltage-clamped fusion pores reports pore size with sub-millisecond time resolution. Preliminary data shows that isolated fusion pores flicker, are long-lived (∼10s) and are of diameter ∼1.5nm.