A molecular brake that modulates spliceosome pausing at detained introns contributes to neurodegeneration

Emerging evidence suggests that intron-detaining transcripts(IDTs)are a nucleus-detained and polyadenylated mRNA pool for cell to quickly and effectively respond to environmental stimuli and stress.However,the underlying mechanisms of detained intron(DI)splicing are still largely unknown.Here,we suggest that post-transcriptional DI splicing is paused at the Bact state,an active spliceosome but not catalytically primed,which depends on Smad Nuclear Interacting Protein 1(SNIP1)and RNPS1(a serine-rich RNA binding protein)interaction.RNPS1 and Bact components preferentially dock at DIs and the RNPS1 docking is sufficient to trigger spliceosome pausing.Haploinsufficiency of Snip1 attenuates neurodegeneration and globally rescues IDT accumulation caused by a previously reported mutant U2 snRNA,a basal spliceosomal component.Snip1 conditional knockout in the cerebellum decreases DI splicing efficiency and causes neurodegeneration.Therefore,we suggest that SNIP1 and RNPS1 form a molecular brake to promote spliceosome pausing,and that its misregulation contributes to neurodegeneration.

The SNW Domain of SKIP Is Required for Its Integration into the Spliceosome and Its Interaction with the Pall Complex in Arabidopsis

SKIP is a conserved protein from yeasts to plants and humans. In plant cells, SKIP is a bifunctional regulator that works in the nucleus as a splicing factor by integrating into the spliceosome and as a transcriptional activator by interacting with the Pall complex. In this study, we identified two nuclear localization signals in SKIP and confirmed that each is sufficient to target SKIP to the nucleus. The SNW domain of SKIP is required for both its function as a splicing factor by promoting integration into the spliceosome in response to stress, and its function as a transcriptional activator by controlling its interaction with the Pall complex to participate in flowering. Truncated proteins that included the SNW domain and the N- or C-terminus of SKIP were still able to carry out the functions of the full-length protein in gene splicing and transcriptional activation in Arabidopsis. In addition, we found that SKIP undergoes 26S proteasome-mediated degrada- tion, and that the C-terminus of SKIP is required to maintain the stability of the protein in plant cells. Together, our findings demonstrate the structural domain organization of SKIP and reveal the core domains and motifs underlying SKIP function in plants.

Emerging roles of spliceosome in cancer and immunity

Precursor messenger RNA(pre-mRNA)splicing is cat-alyzed by an intricate ribonucleoprotein complex called the spliceosome.Although the spliceosome is consid-ered to be general cell"housekeeping"machinery,mutations in core components of the spliceosome fre-quently correlate with cell-or tissue-specific pheno-types and diseases.In this review,we expound the links between spliceosome mutations,aberrant splicing,and human cancers.Remarkably,spliceosome-targeted therapios(STTs)have become efficient anti-cancer strategies for cancer patients with splicing defects.We also highlight the links between spliceosome and immune signaling.Recent studies have shown that some spliceosome gene mutations can result in immune dysregulation and notable phenotypes due to mis-splicing of immune-related genes.Furthermore,several core spliceosome components harbor splicing-inde-pendent immune functions within the cell,expanding the functional repertoire of these diverse proteins.

Spliceosomal GTPase Eftud2 regulates microglial activation and polarization

Elongation factor Tu GTP binding domain protein 2(Eftud2)is a spliceosomal GTPase that serves as an innate immune modulator restricting virus infection.Microglia are the resident innate immune cells and the key players of immune response in the central nervous system.However,the role of Eftud2 in microglia has not been reported.In this study,we performed immunofluorescent staining and western blot assay and found that Eftud2 was upregulated in microglia of a 5xFAD transgenic mouse model of Alzheimer’s disease.Next,we generated an inducible microglia-specific Eftud2 conditional knockout mouse line(CX3CR1-CreER;Eftud2^(f/f) cKO)via Cre/loxP recombination and found that Eftud2 deficiency resulted in abnormal proliferation and promoted anti-inflammatory phenotype activation of microglia.Furthermore,we knocked down Eftud2 in BV2 microglia with siRNA specifically targeting Eftud2 and found that Eftud2-mediated regulation of microglial proinflammatory/anti-inflammatory phenotype activation in response to inflammation might be dependent on the NF-κB signaling pathway.Our findings suggest that Eftud2 plays a key role in regulating microglial polarization and homeostasis possibly through the NF-κB signaling pathway.

A phylogenetic study of <i>Drosophila</i>splicing assembly chaperone RNP-4F associated U4-/U6-snRNA secondary structure

The rnp-4f gene in Drosophila melanogaster encodes nuclear protein RNP-4F. This encoded protein is represented by homologs in other eukaryotic species, where it has been shown to function as an intron splicing assembly factor. Here, RNP-4F is believed to initially bind to a recognition sequence on U6-snRNA, serving as a chaperone to facilitate its association with U4-snRNA by intermolecular hydrogen bonding. RNA conformations are a key factor in spliceosome function, so that elucidation of changing secondary structures for interacting snRNAs is a subject of considerable interest and importance. Among the five snRNAs which participate in removal of spliceosomal introns, there is a growing consensus that U6-snRNA is the most structurally dynamic and may constitute the catalytic core. Previous studies by others have generated potential secondary structures for free U4-and U6-snRNAs, including the Y-shaped U4-/U6-snRNA model. These models were based on study of RNAs from relatively few species, and the popular Y-shaped model remains to be systematically re-examined with reference to the many new sequences generated by recent genomic sequencing projects. We have utilized a comparative phylogenetic approach on 60 diverse eukaryotic species, which resulted in a revised and improved U4-/U6-snRNA secondary structure. This general model is supported by observation of abundant compensatory base mutations in every stem, and incorporates more of the nucleotides into base-paired associations than in previous models, thus being more energetically stable. We have extensively sampled the eukaryotic phylogenetic tree to its deepest roots, but did not find genes potentially encoding either U4-or U6-snRNA in the Giardia and Trichomonas data-bases. Our results support the hypothesis that nuclear introns in these most deeply rooted eukaryotes may represent evolutionary intermediates, sharing characteristics of both group II and spliceosomal introns. An unexpected result of this study was discovery of a potential competitive binding site for Drosophila splicing assembly factor RNP-4Fto a5’-UTR regulatory region within its own pre-mRNA, which may play a role in negative feedback control.

The Sm core protein SmEb regulates salt stress responses through maintaining proper splicing of RCD1 pre-mRNA in Arabidopsis

Salt stress adversely impacts crop production.Several spliceosome components have been implicated in regulating salt stress responses in plants,however,the underlying molecular basis is still unclear.Here we report that the spliceosomal core protein SmEb is essential to salt tolerance in Arabidopsis.Transcriptome analysis showed that SmEb modulates alternative splicing of hundreds of pre-mRNAs in plant response to salt stress.Further study revealed that SmEb is crucial in maintaining proper ratio of two RCD1 splicing variants(RCD1.1/RCD1.2)important for salt stress response.In addition,RCD1.1 but not RCD1.2 is able to interact with the stress regulators and attenuates saltsensitivity by decreasing salt-induced cell death in smeb-1 mutant.Together,our findings uncovered the essential role of SmEb in the regulation of alternative pre-mRNA splicing in salt stress response.

Roles of alternative splicing in infectious diseases: from hosts, pathogens to their interactions

Alternative splicing(AS)is an evolutionarily conserved mechanism that removes introns and ligates exons to generate mature messenger RNAs(mRNAs),extremely improving the richness of transcriptome and proteome.Both mammal hosts and pathogens require AS to maintain their life activities,and inherent physiological heterogeneity between mammals and pathogens makes them adopt different ways to perform AS.Mammals and fungi conduct a two-step transesterification reaction by spliceosomes to splice each individual mRNA(named cis-splicing).Parasites also use spliceosomes to splice,but this splicing can occur among different mRNAs(named trans-splicing).Bacteria and viruses directly hijack the host’s splicing machinery to accomplish this process.Infection-related changes are reflected in the spliceosome behaviors and the characteristics of various splicing regulators(abundance,modification,distribution,movement speed,and conformation),which further radiate to alterations in the global splicing profiles.Genes with splicing changes are enriched in immune-,growth-,or metabolism-related pathways,highlighting approaches through which hosts crosstalk with pathogens.Based on these infection-specific regulators or AS events,several targeted agents have been developed to fight against pathogens.Here,we summarized recent findings in the field of infection-related splicing,including splicing mechanisms of pathogens and hosts,splicing regulation and aberrant AS events,as well as emerging targeted drugs.We aimed to systemically decode host–pathogen interactions from a perspective of splicing.We further discussed the current strategies of drug development,detection methods,analysis algorithms,and database construction,facilitating the annotation of infection-related splicing and the integration of AS with disease phenotype.

The spliceophilin CYP18-2 is mainly involved in the splicing of retained introns under heat stress in Arabidopsis

Peptidyl-prolyl isomerase-like 1(PPIL1)is associated with the human spliceosome complex.However,its function in pre-mRNA splicing remains unclear.In this study,we show that Arabidopsis thaliana CYCLOPHILIN 18-2(AtCYP18-2),a PPIL1 homolog,plays an essential role in heat tolerance by regulating pre-mRNA splicing.Under heat stress conditions,AtCYP18-2 expression was upregulated in mature plants and GFP-tagged AtCYP18-2 redistributed to nuclear and cytoplasmic puncta.We determined that AtCYP18-2 interacts with several spliceosome complex B^(ACT)components in nuclear puncta and is primarily associated with the small nuclear RNAs U5 and U6 in response to heat stress.The AtCYP18-2 loss-of-function allele cyp18-2 engineered by CRISPR/Cas9-mediated gene editing exhibited a hypersensitive phenotype to heat stress relative to the wild type.Moreover,global transcriptome profiling showed that the cyp18-2 mutation affects alternative splicing of heat stress–responsive genes under heat stress conditions,particularly intron retention(IR).The abundance of most intron-containing transcripts of a subset of genes essential for thermotolerance decreased in cyp18-2 compared to the wild type.Furthermore,the intron-containing transcripts of two heat stress-related genes,HEAT SHOCK PROTEIN 101(HSP101)and HEAT SHOCK FACTOR A2(HSFA2),produced functional proteins.HSP101-IR-GFP localization was responsive to heat stress,and HSFA2-Ⅲ-IR interacted with HSF1 and HSP90.1 in plant cells.Our findings reveal that CYP18-2 functions as a splicing factor within the B~(ACT)spliceosome complex and is crucial for ensuring the production of adequate levels of alternatively spliced transcripts to enhance thermotolerance.

Pseudouridines in spliceosomal snRNAs

Spliceosomal RNAs are a family of small nuclear RNAs(snRNAs)that are essential for pre-mRNA splicing.All vertebrate spliceosomal snRNAs are extensively pseudouridylated after transcription.Pseudouridines in spliceosomal snRNAs are generally clustered in regions that are functionally important during splicing.Many of these modified nucleotides are conserved across species lines.Recent studies have demonstrated that spliceosomal snRNA pseudouridylation is catalyzed by two different mechanisms:an RNA-dependent mechanism and an RNA-independent mechanism.The functions of the pseudouridines in spliceosomal snRNAs(U2 snRNA in particular)have also been extensively studied.Experimental data indicate that virtually all pseudouridines in U2 snRNA are functionally important.Besides the currently known pseudouridines(constitutive modifications),recent work has also indicated that pseudouridylation can be induced at novel positions under stress conditions,thus strongly suggesting that pseudouridylation is also a regulatory modification.

Expression of fungal biosynthetic gene clusters in S. cerevisiae for natural product discovery

Fungi are well known for production of antibiotics and other bioactive secondary metabolites,that can be served as pharmaceuticals,therapeutic agents and industrially useful compounds.However,compared with the characterization of prokaryotic biosynthetic gene clusters(BGCs),less attention has been paid to evaluate fungal BGCs.This is partially because heterologous expression of eukaryotic gene constructs often requires replacement of original promoters and terminators,as well as removal of intron sequences,and this substantially slow down the workflow in natural product discovery.It is therefore of interest to investigate the possibility and effectiveness of heterologous expression and library screening of intact BGCs without refactoring in industrial friendly microbial cell factories,such as the yeast Saccharomyces cerevisiae.Here,we discuss the importance of developing new research directions on library screening of fungal BGCs in yeast without refactoring,followed by outlooking prominent opportunities and challenges for future advancement.

Spliceosomal genes in the D.discoideum genome:a comparison with those in H.sapiens,D.melanogaster,A.thaliana and S.cerevisiae

Little is known about pre-mRNA splicing in Dictyostelium discoideum although its genome has been completely sequenced.Our analysis suggests that pre-mRNA splicing plays an important role in D.discoideum gene expression as two thirds of its genes contain at least one intron.Ongoing curation of the genome to date has revealed 40 genes in D.discoideum with clear evidence of alternative splicing,supporting the existence of alternative splicing in this unicellular organism.We identified 160 candidate U2-type spliceosomal proteins and related factors in D.discoideum based on 264 known human genes involved in splicing.Spliceosomal small ribonucleoproteins(snRNPs),PRP19 complex proteins and late-acting proteins are highly conserved in D.discoideum and throughout the metazoa.In non-snRNP and hnRNP families,D.discoideum orthologs are closer to those in A.thaliana,D.melanogaster and H.sapiens than to their counterparts in S.cerevisiae.Several splicing regulators,including SR proteins and CUGbinding proteins,were found in D.discoideum,but not in yeast.Our comprehensive catalog of spliceosomal proteins provides useful information for future studies of splicing in D.discoideum where the efficient genetic and biochemical manipulation will also further our general understanding of pre-mRNA splicing.