snRNP Complex Research Articles

RNA-mediated displacement of an inhibitory snRNP complex activates transcription elongation

The transition from transcription initiation to elongation at the HIV-1 promoter is controlled by Tat, which recruits P-TEFb to TAR RNA to phosphorylate RNA polymerase II. It has long been unclear why the HIV-1 promoter is incompetent for elongation. We report that P-TEFb is recruited to the promoter in a catalytically inactive state bound to the inhibitory 7SK snRNP, thereby preventing elongation. It also has long been believed that TAR functions to recruit Tat to the promoter, but we find that Tat is recruited to the DNA template before TAR is synthesized. We propose that TAR binds Tat and P-TEFb as it emerges on the nascent transcript, competitively displacing the inhibitory 7SK snRNP and activating the P-TEFb kinase. Recruitment of an inhibitory snRNP complex at an early stage in the transcription cycle provides a new paradigm for controlling gene expression with a non-coding RNA.

The More the Merrier

Cell Biology![Figure][1] Cajal bodies in the zebrafish embryo. CREDIT: MAGDALENA STRZELECKA Many biological processes are compartmentalized within the cell by restricting the localization of proteins and other molecules. The spatial organization of these compartments, often bounded by internal membranes, can facilitate the delicate control of complex processes—for instance, the transcription of DNA into RNA, which occurs within the nucleus of eukaryotic cells. However, many non—membrane-bound intracellular compartments have known molecular compositions yet largely unknown functions. Studies attempting to identify their function by mutating or removing protein components to disrupt the compartment physically have been hard to interpret because of the apparent lack of phenotypic changes. This has led to speculation that this kind of compartmentalization may serve to increase the efficiency of an otherwise ongoing process, which may only become rate-limiting under rare physiological conditions. Strzelecka et al. have uncovered just such a critical role for coilin, which is a conserved component of Cajal bodies, during embryogenesis in zebrafish. Cajal bodies are 0.5- to 1-µm structures first described by Ramon y Cajal in 1903. They are found in the nuclei of plant and animal cells, most noticeably in those cells engaging intensely in transcription, and are composed of proteins and RNAs, such as the small nuclear ribonucleoprotein (snRNP) complexes that are important for pre-mRNA splicing. The snRNPs are complicated assemblies that undergo a multistep maturation process, which, it has been proposed, takes place in Cajal bodies. The authors depleted coilin in zebrafish embryos, which caused a disruption of Cajal bodies and the dispersal of snRNPs, before leading subsequently to cell death. These coilin morphants exhibited defective snRNP biogenesis and pre-mRNA splicing, which could be rescued by adding purified mature human snRNPs, revealing a role for coilin in snRNP biogenesis in the zebrafish embryo. The authors suggest that the formation of Cajal bodies concentrates snRNP components and promotes their faithful interaction, which is critical during early embryogenesis when transcriptional activity, and thus the need for snRNPs, is particularly high. These principles may be generally applicable to other non–membrane-bound subcellular compartments with currently unknown functions. Nat. Struct. Mol. Biol. 17 , 403 (2010). [1]: pending:yes

Fox-2A Modulates Protein 4.1R Exon 16 5' Splice Site Activation.

Abstract 778Erythroid differentiation-induced Fox-2A is an important regulator for the differentiation-specific exon 16 splicing switch in protein 4.1R. Up-regulation of Fox-2A in late erythroid differentiation is critical for inclusion of exon 16, which encodes a portion of the spectrin/actin binding domain vital for maintaining the mechanical stability of red blood cell membranes. Fox-2A exerts its splicing enhancing activity in a motif-dependent manner, binding to UGCAUGs located downstream of exon 16. In this study, we investigated the mechanism by which Fox-2A modulates the expression of exon 16. The excision of introns and the joining of exons depends on the recognition and usage of 5' and 3' splice sites (5' ss and 3' ss, respectively) by the splicing machinery. Exon 16 possesses a relatively strong 3' ss but a weak 5' ss. Mutation of the weak 5' ss (GAGIGTTTGT) to a strong consensus 5' ss (GAGIGTAAGT) led to nearly total exon 16 inclusion. While mutations impairing Fox-2A binding drastically reduced exon 16 inclusion in the presence of the weak 5' ss, no effect on exon 16 inclusion was observed when the strong 5' ss was presented with these mutations. These results suggest that Fox-2A facilitates exon 16 splicing by supporting the weak 5' ss. Early recognition of the 5' ss involves base-pairing interaction with the 5' end of U1 snRNA and stabilization by U1 snRNP. Psoralen-mediated UV cross-linking assays revealed a reduction in U1 snRNA recruitment to the weak 5' ss when Fox-2A binding sites were impaired, suggesting that binding of Fox-2A could promote recruitment and stabilization of U1 snRNP to the weak 5' ss. In support for a role of Fox-2A in modulating the activation of the weak 5' ss by recruiting U1 snRNP, we demonstrated that Fox-2A directly interacts with U1 specific protein U1C in an RNA-independent manner. The C-terminal domain of Fox-2A is responsible for its association with U1C. These data suggest a novel mode for exon 16 5' ss activation in which the binding of Fox-2A to an intronic splicing enhancer element UGCAUG may stabilize the pre-mRNA-U1 snRNP complex through interactions with U1C. These could then result in spliceosome commitment complex formation and exon 16 inclusion. Disclosures:No relevant conflicts of interest to declare.

Autosomal-Dominant Retinitis Pigmentosa Caused by a Mutation in SNRNP200, a Gene Required for Unwinding of U4/U6 snRNAs

Mutations in genes associated with the U4/U6-U5 small nuclear ribonucleoprotein (snRNP) complex of the spliceosome are implicated in autosomal-dominant retinitis pigmentosa (adRP), a group of progressive retinal degenerative disorders leading to visual impairment, loss of visual field, and even blindness. We recently assigned a locus (RP33) for adRP to 2cen-q12.1, a region that harbors the SNRNP200 gene encoding hBrr2, another U4/U6-U5 snRNP component that is required for unwinding of U4/U6 snRNAs during spliceosome activation and for disassembly of the spliceosome. Here, we report the identification of a missense mutation, c.3260C>T (p.S1087L), in exon 25 of the SNRNP200 gene in an RP33-linked family. The c.3260C>T substitution showed complete cosegregation with the retinitis pigmentosa (RP) phenotype over four generations, but was absent in a panel of 400 controls. The p.S1087L mutation and p.R1090L, another adRP-associated allele, reside in the "ratchet" helix of the first of two Sec63 domains implicated in the directionality and processivity of nucleic acid unwinding. Indeed, marked defects in U4/U6 unwinding, but not U4/U6-U5 snRNP assembly, were observed in budding yeast for the analogous mutations (N1104L and R1107L) of the corresponding Brr2p residues. The linkage of hBrr2 to adRP suggests that the mechanism of pathogenesis for splicing-factor-related RP may fundamentally derive from a defect in hBrr2-dependent RNA unwinding and a consequent defect in spliceosome activation.

Genetic Complementation Results in Augmented Autoantibody Responses to Lupus-Associated Antigens

Lupus-prone female New Zealand Mixed (NZM)2328 mice develop high titers of anti-nuclear and anti-dsDNA autoantibodies. Despite high expression of type I IFNs, these mice do not develop autoantibodies to the small nuclear ribonucleoprotein (snRNP) complex. Thus, additional genetic factors must regulate the generation of anti-snRNP autoantibodies. In contrast, despite much lower expression of type 1 IFNs, the diabetes-prone NOD mice spontaneously make anti-snRNP autoantibodies, albeit at a low incidence. To determine whether combination of high type I IFN response of NZM mice with appropriate susceptibility genes of NOD mice would result in anti-snRNP Ab response, cohorts of (NZM2328 x NOD)F(1) mice were generated and characterized for development of autoimmunity. In comparison with parental strains, the PBMCs from F(1) mice showed intermediate expression of type I IFN-responsive genes and augmented expression of IL-6 transcripts. TLR7 expression was similar in all strains. The F(1) mice had very high incidence and titer of anti-snRNP autoantibodies, anti-nuclear Abs, and anti-dsDNA autoantibodies. The levels of anti-snRNP autoantibody correlated with the expression levels of type I IFN-responsive genes. None of the F(1) mice developed diabetes, and only female mice developed severe renal disease. Our data demonstrate that only in presence of appropriate susceptibility genes, anti-snRNP autoantibodies are induced and type I IFNs amplify this response. A synergy between IL-6 and type I IFNs might be critical for amplifying overall autoantibody responses in systemic lupus erythematosus. In NZM/NOD F(1) mouse, genetic complementation between NZM and NOD genes leads to expression of phenotypes similar to those seen in certain lupus patients.

A Mechanism for Incorporation of Galectin-3 into the Spliceosome through Its Association with U1 snRNP

Previously, we showed that galectin-1 and galectin-3 are redundant pre-mRNA splicing factors associated with the spliceosome throughout the splicing pathway. Here we present evidence for the association of galectin-3 with snRNPs outside of the spliceosome (i.e., in the absence of pre-mRNA splicing substrate). Immunoprecipitation of HeLa nuclear extract with anti-galectin-3 resulted in the coprecipitation of the five spliceosomal snRNAs, core Sm polypeptides, and the U1-specific protein, U1 70K. When nuclear extract was fractionated on glycerol gradients, some galectin-3 molecules cosedimented with snRNP complexes. This cosedimentation represents bona fide galectin-3--snRNP complexes as (i) immunoprecipitation of gradient fractions with anti-galectin-3 yielded several complexes with varying ratios of snRNAs and associated proteins and (ii) the distribution of galectin-3--snRNP complexes was altered when the glycerol gradient was sedimented in the presence of lactose, a galectin ligand. A complex at approximately 10S showed an association of galectin-3 with U1 snRNP that was sensitive to treatment with ribonuclease A. We tested the ability of this U1 snRNP to recognize an exogenous pre-mRNA substrate. Under conditions that assemble early splicing complexes, we found this isolated galectin-3--U1 snRNP particle was sufficient to load galectin-3 onto a pre-mRNA substrate, but not onto a control RNA lacking splice sites. Pretreatment of the U1 snRNP with micrococcal nuclease abolished the assembly of galectin-3 onto this early complex. These data identify galectin-3 as a polypeptide associated with snRNPs in the absence of splicing substrate and describe a mechanism for the assembly of galectin-3 onto the forming spliceosome.

Interpreting a Low Resolution Map of Human U1 snRNP Using Anomalous Scatterers

SummaryWe recently determined the crystal structure of the functional core of human U1 snRNP, consisting of nine proteins and one RNA, based on a 5.5 Å resolution electron density map. At 5–7 Å resolution, α helices and β sheets appear as rods and slabs, respectively, hence it is not possible to determine protein fold de novo. Using inverse beam geometry, accurate anomalous signals were obtained from weakly diffracting and radiation sensitive P1 crystals. We were able to locate anomalous scatterers with positional errors below 2 Å. This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence. This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.

Immunocytochemical study of estrogen receptor activation factor (E-RAF) and the proteins that interact with nuclear estrogen receptor II (nER II) in epithelial endometrial cells, in the presence and in the absence of estradiol

The localization and abundance of the estrogen receptor activation factor (E-RAF) and a small nuclear ribonucleoprotein (snRNP) complex containing three proteins, p32, p55 and p60, which interact with the nuclear estrogen receptor II (nER II), have been studied in rat endometrial epithelial cells by means of immunofluorescence and high resolution quantitative immunocytochemistry. In the cytoplasm E-RAF is associated with the rough endoplasmic reticulum. In the nucleus it is mainly localized at the interchromatin space, and surrounding the clumps of compact or semi-condensed chromatin. Quantitative analyses show that the abundance of E-RAF in the nucleus increases after ovariectomy and decreases 3 minutes after estradiol administration. These results are in agreement with the currently available biochemical data. Double immunolocalizations demonstrate that p32, p55, p60 co-localize with other splicing-related protein. High resolution immunolocalization shows that p32, p55, p60 are associated with perichromatin fibrils (co-transcriptional splicing) and with clusters of interchromatin granules (storage of splicing-related molecules). The nuclear abundance of the snRNP complex decreases with ovariectomy, increases within 3 minutes after estradiol administration and remains higher than that in ovariectomized animals for 27 minutes. These results strongly support the previous data on the role of nER-II in the regulation of mRNA transcription and its export from the nucleus to the cytoplasm.

CUGBP2 directly interacts with U2 17S snRNP components and promotes U2 snRNA binding to cardiac troponin T pre-mRNA

CUGBP2 (ETR-3/NAPOR/BRUNOL3) promotes inclusion of cardiac troponin T (cTNT) exon 5 via binding between positions 21 and 74 of the downstream intron. The molecular mechanism by which CUGBP2 activates cTNT exon 5 inclusion is unknown. Our results suggest that CUGBP2 promotes exon inclusion by a novel mechanism in which CUGBP2 directly interacts with components of the activated U2 snRNP and enhances binding of U2 snRNP to the branch site located upstream of the exon. Using an in vitro splicing assay, we show that recombinant CUGBP2 enhances complex A formation of a cTNT pre-mRNA. Enhanced complex A assembly requires both the upstream and downstream introns consistent with dual requirements for the downstream CUGBP2-binding site and an upstream branch site for U2 snRNP binding. We also show that CUGBP2 enhances binding of U2 snRNA to the cTNT pre-mRNA consistent with enhanced complex A assembly. Purification of CUGBP2-interacting proteins using tandem affinity purification leads to the demonstration that the core 17S U2 snRNP components, SF3b145 and SF3b49 bind directly to CUGBP2. We conclude that CUGBP2 activates exon inclusion by forming direct interactions with components of the 17S snRNP complex and recruits and/or stabilizes binding of U2 snRNP.

Role of Survival Motor Neuron Complex Components in Small Nuclear Ribonucleoprotein Assembly

Survival motor neuron (SMN) complex is essential for the biogenesis of the small nuclear ribonucleoprotein (snRNP) complex, although the complete role of each SMN complex component for the snRNP synthesis is largely unclear. We have identified an interaction between the two components Gemin2-Gemin7 using the mammalian two-hybrid system. In vitro stability assay revealed that the known SMN-Gemin7 interaction becomes stable in the presence of Gemin2 possibly via the identified Gemin2-Gemin7 interaction. Gemin7 knockdown revealed a decrease in snRNP assembly activity and a decrease in SmE protein, a component of snRNP, in the SMN complex, which was consistent with a previous discussion that the Gemin6-Gemin7 heterodimer may serve as a surrogate for the SmD3-SmB particle in forming a subcore, the intermediate complex for snRNP. Interestingly, we found that Unrip, but not Gemin8, can remove Gemin7 from the stable SMN-Gemin2-Gemin7 ternary complex. In an in vitro snRNP assembly assay using the Unrip knockdown and the untreated cell lysates, we revealed that there was a decrease in Gemin7 and increase in SmB/B' in the SMN complex observed in untreated cells during the assay, suggesting that the Gemin6-Gemin7 heterodimer in the subcore is exchanged by the SmD3-SmB particle to form snRNP. Surprisingly, these changes were not observed in the assay using the Unrip knockdown cell extracts, indicating the importance of Unrip in the formation of snRNP likely via removal of the Gemin6-Gemin7 from the SMN complex. Taken together, these results indicate that snRNP is synthesized by harmonization of the SMN complex components.

Camptothecin releases P-TEFb from the inactive 7SK snRNP complex

An immediate effect of DNA Topoisomerase I inhibitors camptothecin (CPT) and its derivates is the inhibition of transcription. These fast-acting drugs are believed to inhibit transcription by blocking topoisomerase–mediated relief of DNA supercoiling that occurs during transcription elongation. The CPT effects are commonly considered to be due to a collision between the drug-trapped enzyme on the DNA template and the elongating RNAPII. Here we present evidences that CPT treatment induces an early affect on the positive elongation factor b (P-TEFb). The P-TEFb activity is tightly and dynamically regulated, and a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. We found that, shortly after treatment, CPT disrupts the large inactive P-TEFB complex, and such effect is reversible and independent from DNA replication. Thus, CPT modulates P-TEFb equilibrium in a manner similar to Flavopiridol (FP), a pan-Cdk inhibitor proposed as chemotherapeutic agents against cancers. We determined that while FP inhibits Cdk9 leading to hypo-phosphorylation of RNA polymerase II, CPT-mediated release of free P-TEFb correlates with a concomitant hyper-phosphorylation of RNAPII, which in turn alters the levels and distribution of the RNAPII along transcribed genes. The findings that CPT affects P-TEFb activity provide a direct evidence of the mechanism of this drug to inhibit transcription.

Differential 3′ splice site recognition of SMN1 and SMN2 transcripts by U2AF and U2 snRNP

Spinal Muscular atrophy is a prevalent genetic disease caused by mutation of the SMN1 gene, which encodes the SMN protein involved in assembly of small nuclear ribonucleoprotein (snRNP) complexes. A paralog of the gene, SMN2, cannot provide adequate levels of functional SMN because exon 7 is skipped in a significant fraction of the mature transcripts. A C to T transition located at position 6 of exon 7 is critical for the difference in exon skipping between SMN1 and SMN2. Here we report that this nucleotide difference results in increased ultraviolet light-mediated crosslinking of the splicing factor U2AF(65) with the 3' splice site of SMN1 intron 6 in HeLa nuclear extract. U2 snRNP association, analyzed by native gel electrophoresis, is also more efficient on SMN1 than on SMN2, particularly under conditions of competition, suggesting more effective use of limiting factors. Two trans-acting factors implicated in SMN regulation, SF2/ASF and hnRNP A1, promote and repress, respectively, U2 snRNP recruitment to both RNAs. Interestingly, depending on the transcript and the regulatory factor, the effects on U2 binding not always correlate with changes in U2AF(65) crosslinking. Furthermore, blocking recognition of a Tra2-beta1-dependent splicing enhancer located in exon 7 inhibits U2 snRNP recruitment without affecting U2AF(65) crosslinking. Collectively, the results suggest that both U2AF binding and other steps of U2 snRNP recruitment can be control points in SMN splicing regulation.

A survey of nematode SmY RNAs

SmY RNAs are a family of ~70–90 nt small nuclear RNAs found in nematodes. In C. elegans, SmY RNAs copurify in a small ribonucleoprotein (snRNP) complex related to the SL1 and SL2 snRNPs that are involved in nematode mRNA trans-splicing. Here we describe a comprehensive computational analysis of SmY RNA homologs found in the currently available genome sequences. We identify homologs in all sequenced nematode genomes in class Chromadorea. We are unable to identify homologs in a more distantly related nematode species, Trichinella spiralis (class: Dorylaimia), and in representatives of non-nematode phyla that use trans-splicing. Using comparative RNA sequence analysis, we infer a conserved consensus SmY RNA secondary structure consisting of two stems flanking a consensus Sm protein binding site. A representative seed alignment of the SmY RNA family, annotated with the inferred consensus secondary structure, has been deposited with the Rfam RNA families database.

Is Good Housekeeping the Key to Motor Neuron Survival?

Spinal muscular atrophy (SMA) is caused by a drastic reduction in the ubiquitously expressed SMN protein, which is critical for the correct assembly of the snRNP complexes required for RNA splicing. However, it is unclear why loss of SMN and altered snRNP assembly only seem to affect motor neurons. Reporting in this issue, Zhang et al. (2008) challenge prior assumptions about the housekeeping function of SMN and demonstrate that loss of SMN leads to highly tissue-specific effects on splicing.

Dynamic Regulation of ARGONAUTE4 within Multiple Nuclear Bodies in Arabidopsis thaliana

DNA methylation directed by 24-nucleotide small RNAs involves the small RNA-binding protein ARGONAUTE4 (AGO4), and it was previously shown that AGO4 localizes to nucleolus-adjacent Cajal bodies, sites of snRNP complex maturation. Here we demonstrate that AGO4 also localizes to a second class of nuclear bodies, called AB-bodies, which are found immediately adjacent to condensed 45S ribosomal DNA (rDNA) sequences. AB-bodies also contain other proteins involved in RNA-directed DNA methylation including NRPD1b (a subunit of the RNA Polymerase IV complex, RNA PolIV), NRPD2 (a second subunit of this complex), and the DNA methyltransferase DRM2. These two classes of AGO4 bodies are structurally independent—disruption of one class does not affect the other—suggesting a dynamic regulation of AGO4 within two distinct nuclear compartments in Arabidopsis. Abolishing Cajal body formation in a coilin mutant reduced overall AGO4 protein levels, and coilin dicer-like3 double mutants showed a small decrease in DNA methylation beyond that seen in dicer-like3 single mutants, suggesting that Cajal bodies are required for a fully functioning DNA methylation system in Arabidopsis.

The SMN binding protein gemin2 is not involved in motor axon outgrowth

A paramount question in spinal muscular atrophy (SMA) research is why reduced levels of SMN, a ubiquitously expressed protein, leads to a motoneuron-specific disease. It has been hypothesized that SMN may have a dual function: a role in snRNP assembly and a novel function that affects axons. We have previously shown that decreasing Smn levels in zebrafish causes defects in motor axon outgrowth. To determine whether decreasing other components of the snRNP complex would also cause motor axon defects, we knocked down Gemin2, a SMN binding protein involved in snRNP assembly. Moderate knockdown of Gemin2 yields a large percentage of morphologically abnormal embryos with shortened trunks and overall delayed development. Examination of motor axons revealed that only embryos with abnormal body morphology had aberrant motor axons indicating that the motor axon defects are secondary to the overall body defects observed in these embryos. To directly test this, we knocked down Gemin2 specifically in motoneurons using two separate approaches and found that motor axons developed normally. Furthermore, wild-type neurons transplanted into morphologically abnormal gemin2 morphants had aberrant motor axons indicating that the motor axon defects observed when Gemin2 is decreased are secondary to the defects in body morphology. These data show that reduction of Gemin2, unlike reduction of SMN, in zebrafish embryos does not directly cause motor axon outgrowth defects. Since Gemin2 and SMN both function in snRNP biogenesis yet only SMN knockdown causes motor axon defects, these data are consistent with an additional role for SMN that is snRNP independent.

Prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast

Prp8 protein (Prp8p) is a highly conserved pre-mRNA splicing factor and a component of spliceosomal U5 small nuclear ribonucleoproteins (snRNPs). Although it is ubiquitously expressed, mutations in the C terminus of human Prp8p cause the retina-specific disease retinitis pigmentosa (RP). The biogenesis of U5 snRNPs is poorly characterized. We present evidence for a cytoplasmic precursor U5 snRNP in yeast that lacks the mature U5 snRNP component Brr2p and depends on a nuclear localization signal in Prp8p for its efficient nuclear import. The association of Brr2p with the U5 snRNP occurs within the nucleus. RP mutations in Prp8p in yeast result in nuclear accumulation of the precursor U5 snRNP, apparently as a consequence of disrupting the interaction of Prp8p with Brr2p. We therefore propose a novel assembly pathway for U5 snRNP complexes that is disrupted by mutations that cause human RP.

Mutation in the splicing factor Hprp3p linked to retinitis pigmentosa impairs interactions within the U4/U6 snRNP complex

Mutations in PRPF3, a gene encoding the essential pre-mRNA splicing factor Hprp3p, have been identified in patients with autosomal dominant retinitis pigmentosa type 18 (RP18). Patients with RP18 have one of two single amino acid substitutions, Pro493Ser or Thr494Met, at the highly conserved Hprp3p C-terminal region. Pro493Ser occurs sporadically, whereas Thr494Met is observed in several unlinked RP families worldwide. The latter mutation also alters a potential recognition motif for phosphorylation by casein kinase II (CKII). To understand the molecular basis of RP18, we examined the consequences of Thr494Met mutation on Hprp3p molecular interactions with components of the U4/U6.U5 small nuclear ribonucleoprotein particles (snRNPs) complex. Since numerous mutations causing human diseases change pre-mRNA splice sites, we investigated whether Thr494Met substitution affects the processing of PRPF3 mRNA. We found that Thr494Met does not affect PRPF3 mRNA processing, indicating that the mutation may exert its effect primarily at the protein level. We used small hairpin RNAs to specifically silence the endogenous PRPF3 while simultaneously expressing HA-tagged Thr494Met. We demonstrated that the C- but not N-terminal region of Hprp3p is indeed phosphorylated by CKII in vitro and in cells. CKII-mediated Hprp3p phosphorylation was significantly reduced by Thr494Met mutation. Consequently, the Hprp3p C-terminal region is rendered partially defective in its association with itself, Hprp4p, and U4/U6 snRNA. Our findings provide new insights into the biology of Hprp3p and suggest that the loss of Hprp3p phosphorylation at Thr494 is a key step for initiating Thr494Met aberrant interactions within U4/U6 snRNP complex and that these are likely linked to the RP18 phenotype.

Structural Basis for RanGTP Independent Entry of Spliceosomal U snRNPs into the Nucleus

The nuclear import of assembled spliceosomal subunits, the uridine-rich small nuclear ribonucleoprotein particles (U snRNPs), is mediated by a nuclear import receptor adaptor couple of importinβ (Impβ) and snurportin1 (SPN1). In contrast to any other characterized active nuclear import, the Impβ/SPN1/U snRNP complex does not require RanGTP for the terminal release from the nuclear basket of the nuclear pore complex (NPC). The crystal structure of Impβ (127–876) in complex with the Impβ-binding (IBB) domain of SPN1 (1–65) at 2.8-Å resolution reveals that Impβ adopts an open conformation, which is unique for a functional Impβ/cargo complex, and rather surprisingly, it resembles the conformation of the Impβ/RanGTP complex. As binding of RanGTP to Impβ usually triggers the release of import complexes from the NPC, we propose that by already mimicking a conformation similar to Impβ/RanGTP the independent dissociation of Impβ/SPN1 from the nuclear basket is energetically aided.

HMGA1a: sequence‐specific RNA‐binding factor causing sporadic Alzheimer's disease‐linked exon skipping of presenilin‐2 pre‐mRNA

Aberrant exon 5 skipping of presenilin-2 (PS2) pre-mRNA produces a deleterious protein isoform PS2V, which is almost exclusively observed in the brains of sporadic Alzheimer's disease patients. PS2V over-expression in vivo enhances susceptibility to various endoplasmic reticulum (ER) stresses and increases production of amyloid-beta peptides. We previously purified and identified high mobility group A protein 1a (HMGA1a) as a trans-acting factor responsible for aberrant exon 5 skipping. Using heterologous pre-mRNAs, here we demonstrate that a specific HMGA1a-binding sequence in exon 5 adjacent to the 5' splice site is necessary for HMGA1a to inactivate the 5' splice site. An aberrant HMGA1a-U1 snRNP complex was detected on the HMGA1a-binding site adjacent to the 5' splice site during the early splicing reaction. A competitor 2'-O-methyl RNA (2'-O-Me RNA) consisting of the HMGA1a-binding sequence markedly repressed exon 5 skipping of PS2 pre-mRNA in vitro and in vivo. Finally, HMGA1a-induced cell death under ER stress was prevented by transfection of the competitor 2'-O-Me RNA. These results provide insights into the molecular basis for PS2V-associated neurodegenerative diseases that are initiated by specific RNA binding of HMGA1a.