The spliceosome removes introns from messenger RNA precursors (pre-mRNA). Decades of biochemistry and genetics combined with recent structural studies of the spliceosome have produced a detailed view of the mechanism of splicing. In this review, we aim to make this mechanism understandable and provide several videos of the spliceosome in action to illustrate the intricate choreography of splicing. The U1 and U2 small nuclear ribonucleoproteins (snRNPs) mark an intron and recruit the U4/U6.U5 tri-snRNP. Transfer of the 5' splice site (5'SS) from U1 to U6 snRNA triggers unwinding of U6 snRNA from U4 snRNA. U6 folds with U2 snRNA into an RNA-based active site that positions the 5'SS at two catalytic metal ions. The branch point (BP) adenosine attacks the 5'SS, producing a free 5' exon. Removal of the BP adenosine from the active site allows the 3'SS to bind, so that the 5' exon attacks the 3'SS to produce mature mRNA and an excised lariat intron. Expected final online publication date for the Annual Review of Biochemistry, Volume 89 is June 22, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Endometrial cancer (EC) is one of the most common gynecologic malignancies. Emerging studies had demonstrated the mutations in genes could serve as diagnostic or prognostic markers for human cancers. In this study, we screened mutated genes in EC and found that the mutations in KIAA1109, CACNA1C, BSN, AKAP13, CELSR2, and HELZ2 were correlated to the overall survival time in patients with EC. Bioinformatics analysis showed KIAA1109 was involved in regulating NIK/NF-kappaB signaling, CACNA1C was found to regulate cell migration and proliferation, BSN was found to regulate Wnt signaling pathway, CELSR2 was involved in regulating cell-cell adhesion, nuclear import, and protein folding, HELZ2 was found to regulate multiple immune related biological processes, and AKAP13 was involved in regulating translation, mRNA nonsense-mediated decay, rRNA processing, translational initiation, and mRNA splicing via spliceosome. The findings provided a novel therapeutic strategy in patients with EC.

How abiotic stresses affect nuclear proteomes and mediate memory effects? Despite the relevance of this question is capital in the present context of climate change, its answer remains unknown for most species. This work defines how Pinus radiata nuclei respond, adapt, remember and learn from heat stress. Seedlings were heat-stressed at 45 ºC in a 10-day-stress and recovery experiment. Nuclear proteins were isolated and quantified by nLC-MS/MS and tissue DNA methylation dynamics studied, and potential acquired memory was analyzed in recovered plants. A second heat-stress experiment was performed to validate stress memory effects and quantify expression of key genes. Specific nuclear heat responsive proteins were identified, and its biological role evaluated employing a systems biology approach. In addition to HSP, several clusters involved in regulation processes, as epigenomic-driven gene regulation, some transcription factors and a variety of RNA-associated functions were discovered. Nuclei exhibited differential proteome profiles across experiment, being notably H2A histone and methyl cycle enzymes accumulated at the recovery step. Thermopriming effect is possibly linked to H2A abundance and the overaccumulation of spliceosome elements in recovered Pinus radiata plants. Results suggest that epigenetic mechanisms are playing a key role in heat stress tolerance and priming mechanisms.

RNA splicing and spliceosome assembly in eukaryotes occur mainly during transcription. However, co-transcriptional splicing has not yet been explored in plants. Here, we built nascent transcriptomes of chromatin RNAs in Arabidopsis thaliana and showed that, nearly all introns undergo co-transcriptional splicing, and this occurs with higher efficiency for introns in protein-coding genes than for noncoding RNAs. Total intron number and intron position are two predominant features that correlate with co-transcriptional splicing efficiency, and introns with alternative 5' or 3' splice sites are less efficiently spliced. Furthermore, we found that trans-acting protein mutations lead to more introns with increased splicing defects in nascent RNAs than in mature RNAs, and that introns with increased splicing defects in mature RNAs are inefficiently spliced at the co-transcriptional level. Our results not only uncovered widespread co-transcriptional splicing in Arabidopsis, but also identified features that may affect or be affected by co-transcriptional splicing efficiency.

Bromodomain and Extra-Terminal inhibitors (BETi) such as OTX015 are active in Acute Myeloid Leukaemias (AML). Their activity on Leukemic Stem Cells (LSCs) is less documented. We interrogated the anti-LSC activity of OTX015 in a niche-like long-term culture in 26 primary AML samples and validated our findings in vivo. OTX015 impaired LSCs in AMLs harbouring Core Binding Factor or KMT2A gene fusions, NPM1 or chromatin/spliceosome genes mutations, but not in those with aneuploidy/TP53 mutations. In four patients, we dissected the transcriptomic footprint of Bet inhibition on LSCs versus blasts. Our results can instruct future clinical trials of BETi in AML.

The U2 small nuclear ribonucleoprotein (snRNP) is an essential component of the spliceosome, the cellular machine responsible for removing introns from precursor mRNAs (pre-mRNAs) in all eukaryotes. U2 is an extraordinarily dynamic splicing factor and the most frequently mutated in cancers. Cryo-electron microscopy (cryo-EM) has transformed our structural and functional understanding of the role of U2 in splicing. In this review, we synthesize these and other data with respect to a view of U2 as an assembly of interconnected functional modules. These modules are organized by the U2 small nuclear RNA (snRNA) for roles in spliceosome assembly, intron substrate recognition, and protein scaffolding. We describe new discoveries regarding the structure of U2 components and how the snRNP undergoes numerous conformational and compositional changes during splicing. We specifically highlight large scale movements of U2 modules as the spliceosome creates and rearranges its active site. U2 serves as a compelling example for how cellular machines can exploit the modular organization and structural plasticity of an RNP.

The timing of flowering is vital for plant reproductive success and is therefore tightly regulated by endogenous and exogenous cues. In summer annual Arabidopsis (Arabidopsis thaliana) accessions, like Columbia-0, rapid flowering is promoted by repression of the floral repressor FLOWERING LOCUS C (FLC). This is through the activity of the autonomous pathway, a group of proteins with diverse functions including RNA 3'-end processing factors, spliceosome components, a transcription elongation factor and chromatin modifiers. These factors function at the FLC locus linking alternative processing of an antisense long non-coding RNA, called COOLAIR, with delivery of a repressive chromatin environment that affects the transcriptional output. The transcriptional output feeds back to influence the chromatin environment, reinforcing and stabilizing that state. This review summarizes our current knowledge of the autonomous pathway and compares it to similar co-transcriptional mechanisms in other organisms.

Spliceosome-mediated mRNA trans-splicing (SMaRT) is a promising strategy for treatment of genetic diseases which cannot be targeted via classical therapy approaches. SMaRT utilizes an exogenous pre-mRNA trans-splicing molecule (PTM) to correct a diseased target pre-mRNA. This process relies on splicing of two separate pre-mRNA molecules in trans creating a mature chimeric mRNA molecule which consists of the protein coding sequence of the PTM as well as the endogenous mRNA. For therapeutic implications, the most critical step in SMaRT is to develop PTMs resulting in a high ratio of trans-splicing to regular cis-splicing.This protocol provides guidelines on how to design PTMs and describes a fast screening assay to test their efficiencies. To elucidate the therapeutic potential of the best candidates in a more native setting, these PTMs are tested further on mini genes.

Circadian clocks regulate multiple physiological processes in the eye, but their requirement for retinal health remains unclear. We previously showed that Drosophila homologs of spliceosome proteins implicated in human retinitis pigmentosa (RP), the most common genetically inherited cause of blindness, have a role in the brain circadian clock. In this study, we report circadian phenotypes in murine models of RP. We found that mice carrying a homozygous H2309P mutation in Pre-mRNA splicing factor 8 (Prpf8) display a lengthened period of the circadian wheel-running activity rhythm. We show also that the daily cycling of circadian gene expression is dampened in the retina of Prpf8-H2309P mice. Surprisingly, molecular rhythms are intact in the eye cup, which includes the retinal pigment epithelium (RPE), even though the RPE is thought to be the primary tissue affected in this form of RP. Downregulation of Prp31, another RNA splicing factor implicated in RP, leads to period lengthening in a human cell culture model. The period of circadian bioluminescence in primary fibroblasts of human RP patients is not significantly altered. Together, these studies link a prominent retinal disorder to circadian deficits, which could contribute to disease pathology.