The mouse multi-organ proteome from infancy to adulthood.

The early-life organ development and maturation shape the fundamental blueprint for later-life phenotype. However, a multi-organ proteome atlas from infancy to adulthood is currently not available. Herein, we present a comprehensive proteomic analysis of ten mouse organs (brain, heart, lung, liver, kidney, spleen, stomach, intestine, muscle and skin) at three crucial developmental stages (1-, 4- and 8-weeks after birth) acquired using data-independent acquisition mass spectrometry. We detect and quantify 11,533 protein groups across the ten organs and obtain 115 age-related differentially expressed protein groups that are co-expressed in all organs from infancy to adulthood. We find that spliceosome proteins prevalently play crucial regulatory roles in the early-life development of multiple organs, and detect organ-specific expression patterns and sexual dimorphism. This multi-organ proteome atlas provides a fundamental resource for understanding the molecular mechanisms underlying early-life organ development and maturation.

Mechanisms and regulation of spliceosome-mediated pre-mRNA splicing in Saccharomyces cerevisiae.

Pre-mRNA splicing, the removal of introns and ligation of flanking exons, is a crucial step in eukaryotic gene expression. The spliceosome, a macromolecular complex made up of five small nuclear RNAs (snRNAs) and dozens of proteins, assembles on introns via a complex pathway before catalyzing the two transesterification reactions necessary for splicing. All of these steps have the potential to be highly regulated to ensure correct mRNA isoform production for proper cellular function. While Saccharomyces cerevisiae (yeast) has a limited set of intron-containing genes, many of these genes are highly expressed, resulting in a large number of transcripts in a cell being spliced. As a result, splicing regulation is of critical importance for yeast. Just as in humans, yeast splicing can be influenced by protein components of the splicing machinery, structures and properties of the pre-mRNA itself, or by the action of trans-acting factors. It is likely that further analysis of the mechanisms and pathways of splicing regulation in yeast can reveal general principles applicable to other eukaryotes. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing.

AtSNU13 modulates pre-mRNA splicing of RBOHD and ALD1 to regulate plant immunity.

Pre-mRNA splicing is a significant step for post-transcriptional modifications and functions in a wide range of physiological processes in plants. Human NHP2L binds to U4 snRNA during spliceosome assembly; it is involved in RNA splicing and mediates the development of human tumors. However, no ortholog has yet been identified in plants. Therefore, we report At4g12600 encoding the ortholog NHP2L protein, and AtSNU13 associates with the component of the spliceosome complex; the atsnu13 mutant showed compromised resistance in disease resistance, indicating that AtSNU13 is a positive regulator of plant immunity. Compared to wild-type plants, the atsnu13 mutation resulted in altered splicing patterns for defense-related genes and decreased expression of defense-related genes, such as RBOHD and ALD1. Further investigation shows that AtSNU13 promotes the interaction between U4/U6.U5 tri-snRNP-specific 27 K and the motif in target mRNAs to regulate the RNA splicing. Our study highlights the role of AtSNU13 in regulating plant immunity by affecting the pre-mRNA splicing of defense-related genes.

The U1-70K and SRSF1 interaction is modulated by phosphorylation during the early stages of spliceosome assembly.

In eukaryotes, pre-mRNA splicing is vital for RNA processing and orchestrated by the spliceosome, whose assembly starts with the interaction between U1-70K and SR proteins. Despite the significance of the U1-70K/SR interaction, the dynamic nature of the complex and the challenges in obtaining soluble U1-70K have impeded a comprehensive understanding of the interaction at the structural level for decades. We overcome the U1-70K solubility issues, enabling us to characterize the interaction between U1-70K and SRSF1, a representative SR protein. We unveil specific interactions: phosphorylated SRSF1 RS with U1-70K BAD1, and SRSF1 RRM1 with U1-70K RRM. The RS/BAD1 interaction plays a dominant role, whereas the interaction between the RRM domains further enhances the stability of the U1-70K/SRSF1 complex. The RRM interaction involves the C-terminal extension of U1-70K RRM and the conserved acid patches on SRSF1 RRM1 that is involved in SRSF1 phase separation. Our circular dichroism spectra reveal that BAD1 adapts an α-helical conformation and RS is intrinsically disordered. Intriguingly, BAD1 undergoes a conformation switch from α-helix to ß-strand and random coil upon RS binding. In addition to the regulatory mechanism via SRSF1 phosphorylation, the U1-70K/SRSF1 interaction is also regulated by U1-70K BAD1 phosphorylation. We find that U1-70K phosphorylation inhibits the U1-70K and SRSF1 interaction. Our structural findings are validated through in vitro splicing assays and in-cell saturated domain scanning using the CRISPR method, providing new insights into the intricate regulatory mechanisms of pre-mRNA splicing.

Targeting the conserved active site of splicing machines with specific and selective small molecule modulators.

The self-splicing group II introns are bacterial and organellar ancestors of the nuclear spliceosome and retro-transposable elements of pharmacological and biotechnological importance. Integrating enzymatic, crystallographic, and simulation studies, we demonstrate how these introns recognize small molecules through their conserved active site. These RNA-binding small molecules selectively inhibit the two steps of splicing by adopting distinctive poses at different stages of catalysis, and by preventing crucial active site conformational changes that are essential for splicing progression. Our data exemplify the enormous power of RNA binders to mechanistically probe vital cellular pathways. Most importantly, by proving that the evolutionarily-conserved RNA core of splicing machines can recognize small molecules specifically, our work provides a solid basis for the rational design of splicing modulators not only against bacterial and organellar introns, but also against the human spliceosome, which is a validated drug target for the treatment of congenital diseases and cancers.

Therapy-induced secretion of spliceosomal components mediates pro-survival crosstalk between ovarian cancer cells.

Ovarian cancer often develops resistance to conventional therapies, hampering their effectiveness. Here, using ex vivo paired ovarian cancer ascites obtained before and after chemotherapy and in vitro therapy-induced secretomes, we show that molecules secreted by ovarian cancer cells upon therapy promote cisplatin resistance and enhance DNA damage repair in recipient cancer cells. Even a short-term incubation of chemonaive ovarian cancer cells with therapy-induced secretomes induces changes resembling those that are observed in chemoresistant patient-derived tumor cells after long-term therapy. Using integrative omics techniques, we find that both ex vivo and in vitro therapy-induced secretomes are enriched with spliceosomal components, which relocalize from the nucleus to the cytoplasm and subsequently into the extracellular vesicles upon treatment. We demonstrate that these molecules substantially contribute to the phenotypic effects of therapy-induced secretomes. Thus, SNU13 and SYNCRIP spliceosomal proteins promote therapy resistance, while the exogenous U12 and U6atac snRNAs stimulate tumor growth. These findings demonstrate the significance of spliceosomal network perturbation during therapy and further highlight that extracellular signaling might be a key factor contributing to the emergence of ovarian cancer therapy resistance.

Acetylation-dependent regulation of core spliceosome modulates hepatocellular carcinoma cassette exons and sensitivity to PARP inhibitors.

Despite the importance of spliceosome core components in cellular processes, their roles in cancer development, including hepatocellular carcinoma (HCC), remain poorly understood. In this study, we uncover a critical role for SmD2, a core component of the spliceosome machinery, in modulating DNA damage in HCC through its impact on BRCA1/FANC cassette exons and expression. Our findings reveal that SmD2 depletion sensitizes HCC cells to PARP inhibitors, expanding the potential therapeutic targets. We also demonstrate that SmD2 acetylation by p300 leads to its degradation, while HDAC2-mediated deacetylation stabilizes SmD2. Importantly, we show that the combination of Romidepsin and Olaparib exhibits significant therapeutic potential in multiple HCC models, highlighting the promise of targeting SmD2 acetylation and HDAC2 inhibition alongside PARP inhibitors for HCC treatment.

An axonemal intron splicing program sustains Plasmodium male development.

Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.

Genes divided according to the relative position of the longest intron show increased representation in different KEGG pathways.

Despite the fact that introns mean an energy and time burden for eukaryotic cells, they play an irreplaceable role in the diversification and regulation of protein production. As a common feature of eukaryotic genomes, it has been reported that in protein-coding genes, the longest intron is usually one of the first introns. The goal of our work was to find a possible difference in the biological function of genes that fulfill this common feature compared to genes that do not. Data on the lengths of all introns in genes were extracted from the genomes of six vertebrates (human, mouse, koala, chicken, zebrafish and fugu) and two other model organisms (nematode worm and arabidopsis). We showed that more than 40% of protein-coding genes have the relative position of the longest intron located in the second or third tertile of all introns. Genes divided according to the relative position of the longest intron were found to be significantly increased in different KEGG pathways. Genes with the longest intron in the first tertile predominate in a range of pathways for amino acid and lipid metabolism, various signaling, cell junctions or ABC transporters. Genes with the longest intron in the second or third tertile show increased representation in pathways associated with the formation and function of the spliceosome and ribosomes. In the two groups of genes defined in this way, we further demonstrated the difference in the length of the longest introns and the distribution of their absolute positions. We also pointed out other characteristics, namely the positive correlation between the length of the longest intron and the sum of the lengths of all other introns in the gene and the preservation of the exact same absolute and relative position of the longest intron between orthologous genes.

Light regulates nuclear detainment of intron-retained transcripts through COP1-spliceosome to modulate photomorphogenesis.

Intron retention (IR) is the most common alternative splicing event in Arabidopsis. An increasing number of studies have demonstrated the major role of IR in gene expression regulation. The impacts of IR on plant growth and development and response to environments remain underexplored. Here, we found that IR functions directly in gene expression regulation on a genome-wide scale through the detainment of intron-retained transcripts (IRTs) in the nucleus. Nuclear-retained IRTs can be kept away from translation through this mechanism. COP1-dependent light modulation of the IRTs of light signaling genes, such as PIF4, RVE1, and ABA3, contribute to seedling morphological development in response to changing light conditions. Furthermore, light-induced IR changes are under the control of the spliceosome, and in part through COP1-dependent ubiquitination and degradation of DCS1, a plant-specific spliceosomal component. Our data suggest that light regulates the activity of the spliceosome and the consequent IRT nucleus detainment to modulate photomorphogenesis through COP1.

Capturing totipotency in human cells through spliceosomal repression.

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.

Meta-analysis towards FSHD reveals misregulation of neuromuscular junction, nuclear envelope, and spliceosome.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common autosomal dominant muscle disorders, yet no cure or amelioration exists. The clinical presentation is diverse, making it difficult to identify the actual driving pathomechanism among many downstream events. To unravel this complexity, we performed a meta-analysis of 13 original omics datasets (in total 171 FSHD and 129 control samples). Our approach confirmed previous findings about the disease pathology and specified them further. We confirmed increased expression of former proposed DUX4 biomarkers, and furthermore impairment of the respiratory chain. Notably, the meta-analysis provides insights about so far not reported pathways, including misregulation of neuromuscular junction protein encoding genes, downregulation of the spliceosome, and extensive alterations of nuclear envelope protein expression. Finally, we developed a publicly available shiny app to provide a platform for researchers who want to search our analysis for genes of interest in the future.

Intron lariat spliceosomes convert lariats to true circles: implications for intron transposition.

Rare, full-length circular intron RNAs distinct from lariats have been reported in several species, but their biogenesis is not understood. We envisioned and tested a hypothesis for their formation using Saccharomyces cerevisiae, documenting full-length and novel processed circular RNAs from multiple introns. Evidence implicates a previously undescribed catalytic activity of the intron lariat spliceosome (ILS) in which the 3'-OH of the lariat tail (with optional trimming and adenylation by the nuclear 3' processing machinery) attacks the branch, joining the intron 3' end to the 5' splice site in a 3'-5' linked circle. Human U2 and U12 spliceosomes produce analogous full-length and processed circles. Postsplicing catalytic activity of the spliceosome may promote intron transposition during eukaryotic genome evolution.

A role for the C. elegans Argonaute protein CSR-1 in small nuclear RNA 3' processing.

The Integrator is a multi-subunit protein complex that catalyzes the maturation of snRNA transcripts via 3' cleavage, a step required for snRNA incorporation with snRNP for spliceosome biogenesis. Here we developed a GFP based in vivo snRNA misprocessing reporter as a readout of Integrator function and performed a genome-wide RNAi screen for Integrator regulators. We found that loss of the Argonaute encoding csr-1 gene resulted in widespread 3' misprocessing of snRNA transcripts that is accompanied by a significant increase in alternative splicing. Loss of the csr-1 gene down-regulates the germline expression of Integrator subunits 4 and 6 and is accompanied by a reduced protein translation efficiency of multiple Integrator catalytic and non-catalytic subunits. Through isoform and motif mutant analysis, we determined that CSR-1's effect on snRNA processing is dependent on its catalytic slicer activity but does not involve the CSR-1a isoform. Moreover, mRNA-sequencing revealed high similarity in the transcriptome profile between csr-1 and Integrator subunit knockdown via RNAi. Together, our findings reveal CSR-1 as a new regulator of the Integrator complex and implicate a novel role of this Argonaute protein in snRNA 3' processing.

Truncating the spliceosomal 'rope protein' Prp45 results in Htz1 dependent phenotypes.

Spliceosome assembly contributes an important but incompletely understood aspect of splicing regulation. Prp45 is a yeast splicing factor which runs as an extended fold through the spliceosome, and which may be important for bringing its components together. We performed a whole genome analysis of the genetic interaction network of the truncated allele of PRP45 (prp45(1-169)) using synthetic genetic array technology and found chromatin remodellers and modifiers as an enriched category. In agreement with related studies, H2A.Z-encoding HTZ1, and the components of SWR1, INO80, and SAGA complexes represented prominent interactors, with htz1 conferring the strongest growth defect. Because the truncation of Prp45 disproportionately affected low copy number transcripts of intron-containing genes, we prepared strains carrying intronless versions of SRB2, VPS75, or HRB1, the most affected cases with transcription-related function. Intron removal from SRB2, but not from the other genes, partly repaired some but not all the growth phenotypes identified in the genetic screen. The interaction of prp45(1-169) and htz1Δ was detectable even in cells with SRB2 intron deleted (srb2Δi). The less truncated variant, prp45(1-330), had a synthetic growth defect with htz1Δ at 16°C, which also persisted in the srb2Δi background. Moreover, htz1Δ enhanced prp45(1-330) dependent pre-mRNA hyper-accumulation of both high and low efficiency splicers, genes ECM33 and COF1, respectively. We conclude that while the expression defects of low expression intron-containing genes contribute to the genetic interactome of prp45(1-169), the genetic interactions between prp45 and htz1 alleles demonstrate the sensitivity of spliceosome assembly, delayed in prp45(1-169), to the chromatin environment.

Cryptococcus neoformans Slu7 ensures nuclear positioning during mitotic progression through RNA splicing.

The position of the nucleus before it divides during mitosis is variable in different budding yeasts. Studies in the pathogenic intron-rich fungus Cryptococcus neoformans reveal that the nucleus moves entirely into the daughter bud before its division. Here, we report functions of a zinc finger motif containing spliceosome protein C. neoformans Slu7 (CnSlu7) in cell cycle progression. The budding yeast and fission yeast homologs of Slu7 have predominant roles for intron 3' splice site definition during pre-mRNA splicing. Using a conditional knockdown strategy, we show CnSlu7 is an essential factor for viability and is required for efficient cell cycle progression with major role during mitosis. Aberrant nuclear migration, including improper positioning of the nucleus as well as the spindle, were frequently observed in cells depleted of CnSlu7. However, cell cycle delays observed due to Slu7 depletion did not activate the Mad2-dependent spindle assembly checkpoint (SAC). Mining of the global transcriptome changes in the Slu7 knockdown strain identified downregulation of transcripts encoding several cell cycle regulators and cytoskeletal factors for nuclear migration, and the splicing of specific introns of these genes was CnSlu7 dependent. To test the importance of splicing activity of CnSlu7 on nuclear migration, we complemented Slu7 knockdown cells with an intron less PAC1 minigene and demonstrated that the nuclear migration defects were significantly rescued. These findings show that CnSlu7 regulates the functions of diverse cell cycle regulators and cytoskeletal components, ensuring timely cell cycle transitions and nuclear division during mitosis.

The debranching enzyme Dbr1 regulates lariat turnover and intron splicing.

The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of a viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5' splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectrometry. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping.

Genome organization around nuclear speckles drives mRNA splicing efficiency.

The nucleus is highly organized, such that factors involved in the transcription and processing of distinct classes of RNA are confined within specific nuclear bodies1,2. One example is the nuclear speckle, which is defined by high concentrations of protein and noncoding RNA regulators of pre-mRNA splicing3. What functional role, if any, speckles might play in the process of mRNA splicing is unclear4,5. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs and higher co-transcriptional splicing levels than genes that are located farther from nuclear speckles. Gene organization around nuclear speckles is dynamic between cell types, and changes in speckle proximity lead to differences in splicing efficiency. Finally, directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Together, our results integrate the long-standing observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a crucial role for dynamic three-dimensional spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.

L(1)10Bb serves as a conservative determinant for soma-germline communications via cellular non-autonomous effects within the testicular stem cell niche.

The testicular stem cell niche is the central regulator of spermatogenesis in Drosophila melanogaster. However, the underlying regulatory mechanisms are unclear. This study demonstrated the crucial role of lethal (1) 10Bb [l(1)10Bb] in regulating the testicular stem cell niche. Dysfunction of l(1)10Bb in early-stage cyst cells led to male fertility disorders and compromised cyst stem cell maintenance. Moreover, the dysfunction of l(1)10Bb in early-stage cyst cells exerted non-autonomous effects on germline stem cell differentiation, independently of hub signals. Notably, our study highlights the rescue of testicular defects through ectopic expression of L(1)10Bb and the human homologous protein BUD31 homolog (BUD31). In addition, l(1)10Bb dysfunction in early-stage cyst cells downregulated the expression of spliceosome subunits in the Sm and the precursor RNA processing complexes. Collectively, our findings established l(1)10Bb as a pivotal factor in the modulation of Drosophila soma-germline communications within the testicular stem cell niche.