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Pre-mRNA splicing takes place in a large ribonucleoprotein complex, the spliceosome, which contains five small nuclear ribonucleoprotein complexes (snRNPs) and over 100 protein factors. There are two types of introns, the major- and the minor-type, that are excised by two different spliceosomes, assembled by two distinct sets of snRNPs: U1, U2, U4, and U6 snRNPs are required for splicing of the major-type introns; U11, U12, U4atac, and U6atac are their corresponding counterparts, involved in minor-type intron splicing. U5 snRNP is common for both spliceosomes. U4 and U6 snRNAs enter the spliceosome in the form of the U4/U6.U5 tri-snRNP, in which they are extensively base-paired. After splicing, U4 and U6 are released as singular snRNPs. In order to restore the tri-snRNP that can participate in another round of splicing, the U4/U6 hybrid must be recycled. The Prp24 protein in the yeast system, and its distant human homologue p110 have been identified as U4/U6 recycling factors and as specific protein components of U6 and U4/U6 snRNPs. This work focuses on the recycling the minor spliceosome and of the minor spliceosomal U4atac/U6atac snRNP. Using a recycling assay coupled to splicing in vitro, p110 was demonstrated to function also as U4atac/U6atac recycling factor. p110 was detected in U6atac snRNP. In contrast to the major U4/U6, however, it is associated only at background levels with U4atac/U6atac snRNP. Interestingly, U4/U6 and U4atac/U6atac hybrids can be also disrupted independently of splicing in vitro. This process may provide a mechanism to control splicing rates in vivo. The sequence elements of U6 and U6atac snRNA, required for interaction with p110 were identified: U6 nucleotides 38 to 57, and U6atac nucleotides 10 to 30 are sufficient for p110 binding. These regions are the most highly conserved between U6 and U6atac. Finally, singular U6 snRNP was purified from HeLa S100 extract. It contained a novel protein, p85 (cDNA GenBank accession AK001239), identified by mass-spectrometric analysis. p85 localizes to the nucleoplasm, and associates not only with U6, but also with U2 snRNA, and 5S and 5.8S rRNAs. This protein is weakly similar to the yeast rRNA processing factor NOP4, which implicates a function of p85 in maturation of U2 and U6 snRNAs.mRNA-Spleißen findet in einem großen Ribonukleoproteinkomplex (RNP) statt, dem Spleißosom, das sich aus 5 kleinen nukleären RNPs (snRNPs) und über 100 Proteinfaktoren zusammensetzt. Es gibt zwei Klassen von Introns, die sogenannten major- und minor-Introns, die von zwei verschiedenen Spleißosomen prozessiert werden. Diese enthalten unterschiedliche Gruppen von snRNPs: U1, U2, U4 und U6 snRNPs werden für das Spleißen von major-Introns benötigt; U11, U12, U4atac und U6atac bilden die funktionsanalogen snRNPs, welche für das Spleissen von minor-Introns essentiell sind. Der U5 snRNP dagegen kommt in beiden Spleissosom-Formen vor. Die U4 und U6 snRNAs werden in das Spleißosom in Form des U4/U6.U5 tri-snRNPs integriert, in welchem sie eine ausgedehnte Basenpaarung aufweisen. Nach Ablauf der Spleissreaktion werden U4 und U6 separat voneinander freigesetzt. Um daraus den tri-snRNP für weitere Runden der Spleißreaktion zu bilden, muß zunächst der U4/U6-Hybrid durch eine Recycling-Reaktion wiederhergestellt werden. Im Hefesystem wurde das Prp24-Protein, im Humansystem dessen verwandtes Protein p110 als U4/U6-Recyclingfaktor identifiziert und als spezifische Proteinkomponente der U6 und U4/U6 snRNPs. Diese Arbeit konzentriert sich auf das Recycling der U4atac und U6atac snRNPs. Mit Hilfe eines mit der Spleissreaktion gekoppelten in vitro-Assays konnte gezeigt werden, daß p110 auch als U4atac/U6atac Recyclingfaktor wirkt. Das p110-Protein wurde im U6atac snRNP nachgewiesen; im Gegensatz zum normalen U4/U6 snRNP ist p110 nur minimal mit dem U4atac/U6atac snRNP assoziiert. Interessanterweise können die U4/U6- und U4atac/U6atac-Hybride auch unabhängig von der in vitro-Spleissreaktion gespalten werden. Dieser Prozeß stellt in vivo möglicherweise einen Kontrollmechanismus der Spleißreaktion dar. Die für die p110-Interaktion notwendigen Sequenzelemente der U6 und U6atac snRNAs wurden kartiert: p110 benötigt Positionen 38-57 in U6 bzw. 10-30 in U6atac. Diese Bereiche sind zwischen den U6 und U6atac snRNAs am stärksten konserviert. Der singuläre U6 snRNP wurde außerdem biochemisch aus HeLa S100-Extrakt gereinigt. Als eine neue Komponente wurde ein Protein, p85, mittels Massenspektrometrie identifiziert (cDNA GenBank accession AK001239). Diese Protein befindet sich im Nukleoplasma und liegt nicht nur mit U6 snRNA gebunden vor, sondern auch mit U2 sowie 5S und 5.8S ribosomaler RNA. p85 zeigt eine geringe Ähnlichkeit mit dem an der ribosomalen RNA-Prozessierung beteiligten NOP4-Faktor aus der Hefe, was eine Funktion von p85 bei der Reifung der U2 und U6 snRNAs nahelegt

A protein assembly mediates Xist localization and gene silencing

Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear. The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus and binding diverse proteins to achieve X-chromosome inactivation (XCI). The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off. Here we show that the Xist RNA-binding proteins PTBP1, MATR3, TDP-43 and CELF1 assemble on the multivalent E-repeat element of Xist and, via self-aggregation and heterotypic protein–protein interactions, form a condensate in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase, indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing

Autoregulation of Fox protein expression to produce dominant negative splicing factors

The Fox proteins are a family of regulators that control the alternative splicing of many exons in neurons, muscle, and other tissues. Each of the three mammalian paralogs, Fox-1 (A2BP1), Fox-2 (RBM9), and Fox-3 (HRNBP3), produces proteins with a single RNA-binding domain (RRM) flanked by N- and C-terminal domains that are highly diversified through the use of alternative promoters and alternative splicing patterns. These genes also express protein isoforms lacking the second half of the RRM (FoxΔRRM), due to the skipping of a highly conserved 93-nt exon. Fox binding elements overlap the splice sites of these exons in Fox-1 and Fox-2, and the Fox proteins themselves inhibit exon inclusion. Unlike other cases of splicing autoregulation by RNA-binding proteins, skipping the RRM exon creates an in-frame deletion in the mRNA to produce a stable protein. These FoxΔRRM isoforms expressed from cDNA exhibit highly reduced binding to RNA in vivo. However, we show that they can act as repressors of Fox-dependent splicing, presumably by competing with full-length Fox isoforms for interaction with other splicing factors. Interestingly, the Drosophila Fox homolog contains a nearly identical exon in its RRM domain that also has flanking Fox-binding sites. Thus, rather than autoregulation of splicing controlling the abundance of the regulator, the Fox proteins use a highly conserved mechanism of splicing autoregulation to control production of a dominant negative isoform

Recycling of the U12-Type Spliceosome Requires p110, a Component of the U6atac snRNP

U12-dependent introns are spliced by the so-called minor spliceosome, requiring the U11, U12, and U4atac/U6atac snRNPs in addition to the U5 snRNP. We have recently identified U6-p110 (SART3) as a novel human recycling factor that is related to the yeast splicing factor Prp24. U6-p110 transiently associates with the U6 and U4/U6 snRNPs during the spliceosome cycle, regenerating functional U4/U6 snRNPs from singular U4 and U6 snRNPs. Here we investigated the involvement of U6-p110 in recycling of the U4atac/U6atac snRNP. In contrast to the major U6 and U4/U6 snRNPs, p110 is primarily associated with the U6atac snRNP but is almost undetectable in the U4atac/U6atac snRNP. Since p110 does not occur in U5 snRNA-containing complexes, it appears to be transiently associated with U6atac during the cycle of the minor spliceosome. The p110 binding site was mapped to U6 nucleotides 38 to 57 and U6atac nucleotides 10 to 30, which are highly conserved between these two functionally related snRNAs. With a U12-dependent in vitro splicing system, we demonstrate that p110 is required for recycling of the U4atac/U6atac snRNP

p110, a novel human U6 snRNP protein and U4/U6 snRNP recycling factor

During each spliceosome cycle, the U6 snRNA undergoes extensive structural rearrangements, alternating between singular, U4–U6 and U6–U2 base-paired forms. In Saccharomyces cerevisiae, Prp24 functions as an snRNP recycling factor, reannealing U4 and U6 snRNAs. By database searching, we have identified a Prp24-related human protein previously described as p110(nrb) or SART3. p110 contains in its C-terminal region two RNA recognition motifs (RRMs). The N-terminal two-thirds of p110, for which there is no counterpart in the S.cerevisiae Prp24, carries seven tetratricopeptide repeat (TPR) domains. p110 homologs sharing the same domain structure also exist in several other eukaryotes. p110 is associated with the mammalian U6 and U4/U6 snRNPs, but not with U4/U5/U6 tri-snRNPs nor with spliceosomes. Recom binant p110 binds in vitro specifically to human U6 snRNA, requiring an internal U6 region. Using an in vitro recycling assay, we demonstrate that p110 functions in the reassembly of the U4/U6 snRNP. In summary, p110 represents the human ortholog of Prp24, and associates only transiently with U6 and U4/U6 snRNPs during the recycling phase of the spliceosome cycle

The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation.

The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development

Splicing Activation by Rbfox Requires Self-Aggregation through Its Tyrosine-Rich Domain

Proteins of the Rbfox family act with a complex of proteins called the Large Assembly of Splicing Regulators (LASR). We find that Rbfox interacts with LASR via its C-terminal domain (CTD), and this domain is essential for its splicing activity. In addition to LASR recruitment, a low-complexity (LC) sequence within the CTD contains repeated tyrosines that mediate higher-order assembly of Rbfox/LASR and are required for splicing activation by Rbfox. This sequence spontaneously aggregates in solution to form fibrous structures and hydrogels, suggesting an assembly similar to the insoluble cellular inclusions formed by FUS and other proteins in neurologic disease. Unlike the pathological aggregates, we find that assembly of the Rbfox CTD plays an essential role in its normal splicing function. Rather than simple recruitment of individual regulators to a target exon, alternative splicing choices also depend on the higher-order assembly of these regulators within the nucleus

The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation

Abstract The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development

The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain.

The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts, but its role in neuronal physiology is not clear. We show here that central nervous system-specific deletion of the gene encoding Rbfox1 results in heightened susceptibility to spontaneous and kainic acid-induced seizures. Electrophysiological recording revealed a corresponding increase in neuronal excitability in the dentate gyrus of the knockout mice. Whole-transcriptome analyses identified multiple splicing changes in the Rbfox1(-/-) brain with few changes in overall transcript abundance. These splicing changes alter proteins that mediate synaptic transmission and membrane excitation. Thus, Rbfox1 directs a genetic program required in the prevention of neuronal hyperexcitation and seizures. The Rbfox1 knockout mice provide a new model to study the post-transcriptional regulation of synaptic function