scholarly journals Novel Splicing Factor RBM25 Modulates Bcl-x Pre-mRNA 5′ Splice Site Selection

2008 ◽  
Vol 28 (19) ◽  
pp. 5924-5936 ◽  
Author(s):  
AnYu Zhou ◽  
Alexander C. Ou ◽  
Aeri Cho ◽  
Edward J. Benz ◽  
Shu-Ching Huang
Keyword(s):  
Splice Site ◽  
Dependent Manner ◽  
Splice Site Selection ◽  
Exon 2 ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp ◽  
Nuclear Ribonucleoprotein ◽  
Dose Dependent ◽  
Selection Of

ABSTRACT RBM25 has been shown to associate with splicing cofactors SRm160/300 and assembled splicing complexes, but little is known about its splicing regulation. Here, we characterize the functional role of RBM25 in alternative pre-mRNA splicing. Increased RBM25 expression correlated with increased apoptosis and specifically affected the expression of Bcl-x isoforms. RBM25 stimulated proapoptotic Bcl-xS 5′ splice site (5′ ss) selection in a dose-dependent manner, whereas its depletion caused the accumulation of antiapoptotic Bcl-xL. Furthermore, RBM25 specifically bound to Bcl-x RNA through a CGGGCA sequence located within exon 2. Mutation in this element abolished the ability of RBM25 to enhance Bcl-xS 5′ ss selection, leading to decreased Bcl-xS isoform expression. Binding of RBM25 was shown to promote the recruitment of the U1 small nuclear ribonucleoprotein particle (snRNP) to the weak 5′ ss; however, it was not required when a strong consensus 5′ ss was present. In support of a role for RBM25 in modulating the selection of a 5′ ss, we demonstrated that RBM25 associated selectively with the human homolog of yeast U1 snRNP-associated factor hLuc7A. These data suggest a novel mode for Bcl-xS 5′ ss activation in which binding of RBM25 with exonic element CGGGCA may stabilize the pre-mRNA-U1 snRNP through interactions with hLuc7A.

scholarly journals U1 snRNP-Dependent Suppression of Polyadenylation: Physiological Role and Therapeutic Opportunities in Cancer

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Lee Spraggon ◽  
Luca Cartegni
Keyword(s):  
Splice Site ◽  
Physiological Role ◽  
Alternative Polyadenylation ◽  
Mrna Processing ◽  
Splice Site Selection ◽  
General Role ◽  
Global Function ◽  
U1 Snrnp ◽  
Polyadenylation Signals

Pre-mRNA splicing and polyadenylation are critical steps in the maturation of eukaryotic mRNA. U1 snRNP is an essential component of the splicing machinery and participates in splice-site selection and spliceosome assembly by base-pairing to the 5′ splice site. U1 snRNP also plays an additional, nonsplicing global function in 3′ end mRNA processing; it actively suppresses the polyadenylation machinery from using early, mostly intronic polyadenylation signals which would lead to aberrant, truncated mRNAs. Thus, U1 snRNP safeguards pre-mRNA transcripts against premature polyadenylation and contributes to the regulation of alternative polyadenylation. Here, we review the role of U1 snRNP in 3′ end mRNA processing, outline the evidence that led to the recognition of its physiological, general role in inhibiting polyadenylation, and finally highlight the possibility of manipulating this U1 snRNP function for therapeutic purposes in cancer.

scholarly journals A Novel Yeast U2 snRNP Protein, Snu17p, Is Required for the First Catalytic Step of Splicing and for Progression of Spliceosome Assembly

2001 ◽  
Vol 21 (9) ◽  
pp. 3037-3046 ◽  
Author(s):  
Alexander Gottschalk ◽  
Cornelia Bartels ◽  
Gitte Neubauer ◽  
Reinhard Lührmann ◽  
Patrizia Fabrizio
Keyword(s):  
Affinity Purification ◽  
Rna Recognition Motif ◽  
Spliceosome Assembly ◽  
Exon 2 ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp ◽  
Catalytically Active ◽  
Snrnp Protein ◽  
Nuclear Ribonucleoprotein

ABSTRACT We have isolated and microsequenced Snu17p, a novel yeast protein with a predicted molecular mass of 17 kDa that contains an RNA recognition motif. We demonstrate that Snu17p binds specifically to the U2 small nuclear ribonucleoprotein (snRNP) and that it is part of the spliceosome, since the pre-mRNA and the lariat-exon 2 are specifically coprecipitated with Snu17p. Although the SNU17gene is not essential, its knockout leads to a slow-growth phenotype and to a pre-mRNA splicing defect in vivo. In addition, the first step of splicing is dramatically decreased in extracts prepared from thesnu17 deletion (snu17Δ) mutant. This defect is efficiently reversed by the addition of recombinant Snu17p. To investigate the step of spliceosome assembly at which Snu17p acts, we have used nondenaturing gel electrophoresis. In Snu17p-deficient extracts, the spliceosome runs as a single slowly migrating complex. In wild-type extracts, usually at least two distinct complexes are observed: the prespliceosome, or B complex, containing the U2 but not the U1 snRNP, and the catalytically active spliceosome, or A complex, containing the U2, U6, and U5 snRNPs. Northern blot analysis and affinity purification of the snu17Δ spliceosome showed that it contains the U1, U2, U6, U5, and U4 snRNPs. The unexpected stabilization of the U1 snRNP and the lack of dissociation of the U4 snRNP suggest that loss of Snu17p inhibits the progression of spliceosome assembly prior to U1 snRNP release and after [U4/U6.U5] tri-snRNP addition.

scholarly journals The Pre-mRNA 5′ Cap Determines Whether U6 Small Nuclear RNA Succeeds U1 Small Nuclear Ribonucleoprotein Particle at 5′ Splice Sites

1998 ◽  
Vol 18 (12) ◽  
pp. 7510-7520 ◽  
Author(s):  
Laura O’Mullane ◽  
Ian C. Eperon
Keyword(s):  
Splice Site ◽  
Major Effect ◽  
Small Nuclear Rna ◽  
Splice Sites ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrna ◽  
U1 Snrnp ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein

ABSTRACT Efficient splicing of the 5′-most intron of pre-mRNA requires a 5′ m7G(5′)ppp(5′)N cap, which has been implicated in U1 snRNP binding to 5′ splice sites. We demonstrate that the cap alters the kinetic profile of U1 snRNP binding, but its major effect is on U6 snRNA binding. With two alternative wild-type splice sites in an adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the site to which cap-independent U1 snRNP binding is stronger and that is used predominantly in splicing; with two consensus sites, the cap acts on both, even though one is substantially preferred for splicing. However, the most striking quantitative effect of the 5′ cap is neither on U1 snRNP binding nor on the assembly of large complexes but on the replacement of U1 snRNP by U6 snRNA at the 5′ splice site. Inhibition of splicing by a cap analogue is correlated with the loss of U6 interactions at the 5′ splice site and not with any loss of U1 snRNP binding.

scholarly journals Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.

1991 ◽  
Vol 11 (12) ◽  
pp. 5919-5928 ◽  
Author(s):  
P J Grabowski ◽  
F U Nasim ◽  
H C Kuo ◽  
R Burch
Keyword(s):  
Splice Site ◽  
Failure To Rescue ◽  
Ribonucleoprotein Particle ◽  
Upstream Site ◽  
Site Model ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp ◽  
Alternatively Spliced ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to U1 RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.

scholarly journals Importin β-depending Nuclear Import Pathways: Role of the Adapter Proteins in the Docking and Releasing Steps

2003 ◽  
Vol 14 (5) ◽  
pp. 2104-2115 ◽  
Author(s):  
Christiane Rollenhagen ◽  
Petra Mühlhäusser ◽  
Ulrike Kutay ◽  
Nelly Panté
Keyword(s):  
Nuclear Import ◽  
Nuclear Pore ◽  
Adapter Proteins ◽  
Adapter Protein ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp ◽  
Importin Α ◽  
Localization Signal ◽  
Nuclear Ribonucleoprotein

Nuclear imports of uridine-rich small nuclear ribonucleoprotein (U1 snRNP) and proteins with classical nuclear localization signal (cNLS-protein) are mediated by importin β. However, due to the presence of different import signals, the adapter protein of the imported molecules and importin β is different for each pathway. Although the adapter for cNLS-protein is importin α, the adapter for U1 snRNP is snurportin1 (SPN1). Herein, we show that the use of distinct adapters by importin β results in differences at the docking and releasing step for these two import pathways. Nuclear pore complex (NPC) docking of U1 snRNP but not of cNLS-protein was inhibited by an anti-CAN/Nup214 antibody. Thus, the initial NPC-binding site is different for each pathway. Pull-down assays between immobilized SPN1 and two truncated forms of importin β documented that SPN1 and importin α have different binding sites on importin β. Importin β fragment 1–618, which binds to SPN1 but not to importin α, was able to support the nuclear import of U1 snRNPs. After the translocation through the NPC, both import complexes associated with the nuclear side of the NPC. However, we found that the nature of the importin β-binding domain of the adapters influences the release of the cargo into the nucleoplasm.

scholarly journals Role of the Modular Domains of SR Proteins in Subnuclear Localization and Alternative Splicing Specificity

1997 ◽  
Vol 138 (2) ◽  
pp. 225-238 ◽  
Author(s):  
Javier F. Cáceres ◽  
Tom Misteli ◽  
Gavin R. Screaton ◽  
David L. Spector ◽  
Adrian R. Krainer
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Site Selection ◽  
Cellular Localization ◽  
Sr Proteins ◽  
Dependent Manner ◽  
Splice Site Selection ◽  
Subnuclear Localization

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5′ splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.

scholarly journals U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor of 65 kDa, U2AF65, can promote U1 snRNP recruitment to 5′ splice sites

2003 ◽  
Vol 372 (1) ◽  
pp. 235-240 ◽  
Author(s):  
Patrik FÖRCH ◽  
Livia MERENDINO ◽  
Concepción MARTÍNEZ ◽  
Juan VALCÁRCEL
Keyword(s):  
Splice Sites ◽  
Ribonucleoprotein Particle ◽  
Rich Domain ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Nuclear Ribonucleoprotein ◽  
Auxiliary Factor ◽  
Small Nuclear Ribonucleoprotein Particle

The splicing factor U2AF65, U2 small nuclear ribonucleoprotein particle (snRNP) auxillary factor of 65 kDa, binds to pyrimidine-rich sequences at 3′ splice sites to recruit U2 snRNP to pre-mRNAs. We report that U2AF65 can also promote the recruitment of U1 snRNP to weak 5′ splice sites that are followed by uridine-rich sequences. The arginine- and serine-rich domain of U2AF65 is critical for U1 recruitment, and we discuss the role of its RNA–RNA annealing activity in this novel function of U2AF65.

scholarly journals Commitment of yeast pre-mRNA to the splicing pathway requires a novel U1 small nuclear ribonucleoprotein polypeptide, Prp39p

1994 ◽  
Vol 14 (6) ◽  
pp. 3623-3633
Author(s):  
S R Lockhart ◽  
B C Rymond
Keyword(s):  
Splice Site ◽  
Mrna Splicing ◽  
Small Nuclear Ribonucleoprotein ◽  
Cell Extracts ◽  
U1 Snrnp ◽  
Intron Recognition ◽  
Band Characteristic ◽  
Nuclear Ribonucleoprotein ◽  
Temperature Inactivation

The binding of a U1 small nuclear ribonucleoprotein (snRNP) particle to the 5' splice site region of a pre-mRNA is a primary step of intron recognition. In this report, we identify a novel 75-kDa polypeptide of Saccharomyces cerevisiae, Prp39p, necessary for the stable interaction of mRNA precursors with the snRNP components of the pre-mRNA splicing machinery. In vivo, temperature inactivation or metabolic depletion of Prp39p blocks pre-mRNA splicing and causes growth arrest. Analyses of cell extracts reveal a specific and dramatic increase in the electrophoretic mobility of the U1 snRNP particle upon Prp39p depletion and demonstrate that extracts deficient in Prp39p activity are unable to form either the CC1 or CC2 commitment complex band characteristic of productive U1 snRNP/pre-mRNA association. Immunological studies establish that Prp39p is uniquely associated with the U1 snRNP and is recruited with the U1 snRNP into splicing complexes. On the basis of these and related observations, we propose that Prp39p functions, at least in part, prior to stable branch point recognition by the U1 snRNP particle to facilitate or stabilize the U1 snRNP/5' splice site interaction.

scholarly journals Viral neuroinvasion and neurotropism without neuronal damage in the hACE2 mouse model of COVID-19

2021 ◽  
Author(s):  
Frauke Seehusen ◽  
Jordan J. Clark ◽  
Parul Sharma ◽  
Krishanthi Subramaniam ◽  
Sabina Wunderlin Giuliani ◽  
...  
Keyword(s):  
Influenza A ◽  
Immune Cell ◽  
Neuronal Damage ◽  
Cerebrovascular Diseases ◽  
Dependent Manner ◽  
Angiotensin Converting Enzyme 2 ◽  
The Central Nervous System ◽  
Dose Dependent ◽  
Selection Of

AbstractCoronavirus disease 2019 (COVID-19) is a primarily respiratory disease with variable clinical courses for which animal models are needed to gather insights into the pathogenesis of its causative virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), in human patients. SARS-CoV-2 not only affects the respiratory tract but also the central nervous system (CNS), leading to neurological symptoms such as loss of smell and taste, headache, fatigue or severe complications like cerebrovascular diseases. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a well-known model of SARS-CoV-2 infection. In the present study, it served to investigate the spatiotemporal distribution and pathomorphological features in the CNS following intranasal infection with relatively low SARS-CoV-2 doses and after prior influenza A virus infection.In K18-hACE2 mice, SARS-CoV-2 was found to frequently spread to and within the CNS during the later phase (day 7) of infection. Infection was restricted to neurons and appeared to first affect the olfactory bulb and spread from there mainly in basally orientated regions in the brain and into the spinal cord, in a dose dependent manner and independent of ACE2 expression. Neuronal infection was not associated with cell death, axonal damage or demyelination. However, microglial activation, microgliosis and a mild macrophage and T cell dominated inflammatory response was consistently observed. This was accompanied by apoptotic death of endothelial, microglial and immune cells, without evidence of viral infection of glial cells, endothelial cells and leukocytes.Taken together, microgliosis and immune cell apoptosis indicate a potential important role of microglial cells for the pathogenesis and viral effect in COVID-19 and possible impairment of neurological functions, especially in long COVID. These data may also be informative for the selection of therapeutic candidates, and broadly support investigation of agents with adequate penetration into relevant regions of the CNS.

Sign in / Sign up

Export Citation Format

Share Document