Mechanisms of modulation of pre-mRNA 3D structural scaffolds for splicing substrate definition

Coordination of different serine-arginine-rich (SR) proteins - a class of critical splicing activators - facilitates recognition of the highly degenerate cognate splice signal sequences against the background sequences. Yet, the mechanistic details of their actions remain unclear. Here we show that cooperative binding of SR proteins to exonic and intronic motifs remodels the pre-mRNA 3D structural scaffold. The scaffold generated by pre-mRNA-specific combinations of different SR proteins in an appropriate stoichiometry is recognized by U1 snRNP. A large excess of U1 snRNP particles displaces the majority of the bound SR protein molecules from the remodeled pre-mRNA. A higher than optimal stoichiometry of SR proteins occludes the binding sites on the pre-mRNA, raising the U1 snRNP levels required for SR protein displacement and potentially impeding spliceosome assembly. This novel step is important for distinguishing the substrate and the non-substrate by U2AF65 - the primary 3' splice site-recognizing factor. Overall, this work elucidates early regulatory steps of mammalian splicing substrate definition by SR proteins.

Phylogenetic Comparison and Splicing Analysis of the U1 snRNP-specific Protein U1C in Eukaryotes

As a pivotal regulator of 5’ splice site recognition, U1 small nuclear ribonucleoprotein (U1 snRNP)-specific protein C (U1C) regulates pre-mRNA splicing by interacting with other components of the U1 snRNP complex. Previous studies have shown that U1 snRNP and its components are linked to a variety of diseases, including cancer. However, the phylogenetic relationships and expression profiles of U1C have not been studied systematically. To this end, we identified a total of 110 animal U1C genes and compared them to homologues from yeast and plants. Bioinformatics analysis shows that the structure and function of U1C proteins is relatively conserved and is found in multiple copies in a few members of the U1C gene family. Furthermore, the expression patterns reveal that U1Cs have potential roles in cancer progression and human development. In summary, our study presents a comprehensive overview of the animal U1C gene family, which can provide fundamental data and potential cues for further research in deciphering the molecular function of this splicing regulator.

Polyautoimmunity Clusters as a New Taxonomy of Autoimmune Diseases

Polyautoimmunity (PolyA) is an emerging concept that may help to develop a better classification of autoimmune diseases (ADs). Thus, we aimed to develop new taxonomy based on PolyA. Two-hundred and fifty-four consecutive patients were included with rheumatoid arthritis (RA, n:146), systemic lupus erythematosus (SLE, n:45), Sjögrens syndrome (SS, n:29), autoimmune thyroid disease (AITD, n:17) and systemic sclerosis (SSc, n:17). Clinical features, autoantigen array chip, lymphocytes immunophenotype and cytokine profile were assessed simultaneously. The coexistence of two or more ADs with classification criteria was termed Overt PolyA, whereas the presence of autoantibodies unrelated to the index AD, without criteria fulfillment, was named Latent PolyA. Combination of IgG autoantibodies yielded high accuracy for classification of ADs. In SLE, Histone H2A, Sm/RNP, ssDNA, and dsDNA IgG autoantibodies were the most predictive autoantibodies for this condition. Laminin, Ro/SSA (52 kDa), and U1−snRNP B/B for SS; Thyroglobulin for AITD; Ribo Phosphoprotein P1, and CENP-A for SSc. Interestingly, Thyroglobulin and U1−snRNP B/B' were mutual diagnostic biomarkers in SS and SSc. Latent PolyA showed in nearly 70% of patients, whereas overt PolyA was most common in AITD (82.4%) and SLE (40%). Cluster analysis based on autoantibodies yielded three clusters of which clusters 2 and 3 exhibited high frequency of latent and overt PolyA with distinctive clinical and immunological phenotypes. Combination of autoantibodies demonstrated high performance for classification of ADs. Patients with both latent and overt PolyA cluster together and exhibit differential clinical and immunological features. High prevalence of latent and overt PolyA advocates for routinary surveillance in clinical settings.

The upstream 5′ splice site remains associated to the transcription machinery during intron synthesis

In the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF along with other proteins. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns in human cells, we discovered that the nascent 5′ splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. This association functionally corresponds with splicing outcome, involves bona fide 5′ splice sites and cryptic intronic sites, and occurs transcriptome-wide. Overall, our findings reveal that the upstream 5′ splice sites remain attached to the transcriptional machinery during intron synthesis and are thus brought into proximity of the 3′ splice sites; potentially mediating the rapid splicing of long introns.

Phylogenetic comparison and splice site conservation of eukaryotic U1 snRNP-specific U1-70K gene family

Eukaryotic cells can expand their coding ability by using their splicing machinery, spliceosome, to process precursor mRNA (pre-mRNA) into mature messenger RNA. The mega-macromolecular spliceosome contains multiple subcomplexes, referred to as small nuclear ribonucleoproteins (snRNPs). Among these, U1 snRNP and its central component, U1-70K, are crucial for splice site recognition during early spliceosome assembly. The human U1-70K has been linked to several types of human autoimmune and neurodegenerative diseases. However, its phylogenetic relationship has been seldom reported. To this end, we carried out a systemic analysis of 95 animal U1-70K genes and compare these proteins to their yeast and plant counterparts. Analysis of their gene and protein structures, expression patterns and splicing conservation suggest that animal U1-70Ks are conserved in their molecular function, and may play essential role in cancers and juvenile development. In particular, animal U1-70Ks display unique characteristics of single copy number and a splicing isoform with truncated C-terminal, suggesting the specific role of these U1-70Ks in animal kingdom. In summary, our results provide phylogenetic overview of U1-70K gene family in vertebrates. In silico analyses conducted in this work will act as a reference for future functional studies of this crucial U1 splicing factor in animal kingdom.

The U1 snRNP component RBP45d regulates temperature-responsive flowering in Arabidopsis thaliana

Pre-mRNA splicing is a crucial step of gene expression whereby the spliceosome produces constitutively and alternatively spliced transcripts that not only diversify the transcriptome but also play essential functions during plant development and responses to environmental changes. Numerous evidences indicate that regulation at the pre-mRNA splicing step is important for flowering time control, however the components and detailed mechanism underlying this process remain largely unknown. Here, we identified a previously unknown splicing factor in Arabidopsis thaliana, RNA BINDING PROTEIN 45d (RBP45d), a member of the RBP45/47 family. Using sequence comparison and biochemical analysis, we determined that RBP45d is a component of the U1 small nuclear ribonucleoprotein (U1 snRNP) with functions distinct from other family members. RBP45d associates with the U1 snRNP by interacting with pre-mRNA-processing factor 39a (PRP39a) and directly regulates alternative splicing (AS) for a specific set of genes. Plants with loss of RBP45d function exhibit defects in temperature-induced flowering potentially due to the mis-regulation of temperature-sensitive AS by RBP45d and PRP39a of the key flowering gene FLOWERING LOCUS M. Taken together, we report that RBP45d is a novel U1 snRNP component in plants that functions together with PRP39a in temperature-mediated flowering.

ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5' splice site motif within nuclear speckles

Quality control of mRNA represents an important regulatory mechanism for gene expression in eukaryotes. One component of this quality control is the nuclear retention and decay of misprocessed RNAs. Previously, we demonstrated that mature mRNAs containing a 5' splice site (5'SS) motif, which is typically found in misprocessed RNAs such as intronic polyadenylated (IPA) transcripts, are nuclear retained and degraded. Here we demonstrate that these transcripts require the zinc finger protein ZFC3H1 for their decay and nuclear retention into nuclear speckles. Furthermore, we find that U1-70K, a component of the U1 snRNP spliceosomal complex, is also required for their nuclear retention and likely functions in the same pathway as ZFC3H1. Finally, we show that the disassembly of nuclear speckles impairs the nuclear retention of mRNAs with 5'SS motifs. Together, our results suggest a model where mRNAs with 5'SS motifs are recognized by U1 snRNP, which then acts with ZFC3H1 to both promote their decay and prevent nuclear export of these mRNAs by sequestering them in nuclear speckles. Our results highlight a splicing independent role of U1 snRNP and indicate that it works in conjunction with ZFC3H1 in preventing the nuclear export of misprocessed mRNAs.

Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling

Splicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre-mRNA) into mRNA by excising introns and ligating exons. This task is accomplished by the spliceosome, a macromolecular machine that must undergo sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are activated remain obscure. Here we show that Prp28, a yeast DEAD-box ATPase, transiently interacts with the conserved 5′ splice-site (5′SS) GU dinucleotide and makes splicing-dependent contacts with the U1 snRNP protein U1C, and U4/U6.U5 tri-snRNP proteins, Prp8, Brr2, and Snu114. We further show that Prp28’s ATPase activity is potentiated by the phosphorylated Npl3, but not the unphosphorylated Npl3, thus suggesting a strategy for regulating DExD/H-box ATPases. We propose that Npl3 is a functional counterpart of the metazoan-specific Prp28 N-terminal region, which can be phosphorylated and serves as an anchor to human spliceosome.

Multi-Factor Authentication of Potential 5′ Splice Sites by the Saccharomyces cerevisiae U1 snRNP

In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5' splice site (5' SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5' SS recognition have largely been done in vivo or in cellular extracts where the U1/5' SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used co-localization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5' SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5' SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5' SS and preferentially accumulate at these sequences rather than on close cognates.