Characterization and prognostic significance of alternative splicing events in lower‐grade diffuse gliomas

Alternative splicing (AS) is assumed to play important roles in the progression and prognosis of cancer. Currently, the comprehensive analysis and clinical relevance of AS in lower‐grade diffuse gliomas have not been systematically addressed. Here, we gathered alternative splicing data of lower‐grade diffuse gliomas from SpliceSeq. Based on the Percent Spliced In (PSI) values of 515 lower‐grade diffuse glioma patients from the Cancer Genome Atlas (TCGA), we performed subtype‐differential AS analysis and consensus clustering to determine robust clusters of patients. A total of 48 050 AS events in 10 787 genes in lower‐grade diffuse gliomas were profiled. Subtype‐differential splicing analysis and functional annotation revealed that spliced genes were significantly enriched in numerous cancer‐related biological phenotypes and signalling pathways. Consensus clustering using AS events identified three robust clusters of patients with distinguished pathological and prognostic features. Moreover, each cluster was also associated with distinct genomic alterations. Finally, we developed and validated an AS‐related signature with Cox proportional hazards model. The signature, significantly associated with clinical and molecular features, could serve as an independent prognostic factor for lower‐grade diffuse gliomas. Thus, our results indicated that AS events could discriminate molecular subtypes and have prognostic impact in lower‐grade diffuse gliomas.     

Frontotemporal dementia‐linked P112H mutation of TDP‐43 induces protein structural change and impairs its RNA binding function

TDP‐43 forms the primary constituents of the cytoplasmic inclusions contributing to various neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia (FTD). Over 60 TDP‐43 mutations have been identified in patients suffering from these two diseases, but most variations are located in the protein''s disordered C‐terminal glycine‐rich region. P112H mutation of TDP‐43 has been uniquely linked to FTD, and is located in the first RNA recognition motif (RRM1). This mutation is thought to be pathogenic, but its impact on TDP‐43 at the protein level remains unclear. Here, we compare the biochemical and biophysical properties of TDP‐43 truncated proteins with or without P112H mutation. We show that P112H‐mutated TDP‐43 proteins exhibit higher thermal stability, impaired RNA‐binding activity, and a reduced tendency to aggregate relative to wild‐type proteins. Near‐UV CD, 2D‐nuclear‐magnetic resonance, and intrinsic fluorescence spectrometry further reveal that the P112H mutation in RRM1 generates local conformational changes surrounding the mutational site that disrupt the stacking interactions of the W113 side chain with nucleic acids. Together, these results support the notion that P112H mutation of TDP‐43 contributes to FTD through functional impairment of RNA metabolism and/or structural changes that curtail protein clearance.     

Disease‐linked TDP‐43 hyperphosphorylation suppresses TDP‐43 condensation and aggregation

Post‐translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA‐binding protein TAR DNA‐binding protein (TDP‐43), is hyperphosphorylated in disease on several C‐terminal serine residues, a process generally believed to promote TDP‐43 aggregation. Here, we however find that Casein kinase 1δ‐mediated TDP‐43 hyperphosphorylation or C‐terminal phosphomimetic mutations reduce TDP‐43 phase separation and aggregation, and instead render TDP‐43 condensates more liquid‐like and dynamic. Multi‐scale molecular dynamics simulations reveal reduced homotypic interactions of TDP‐43 low‐complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP‐43, but suppress accumulation of TDP‐43 in membrane‐less organelles and promote its solubility in neurons. We speculate that TDP‐43 hyperphosphorylation may be a protective cellular response to counteract TDP‐43 aggregation.     

ATP regulates RNA‐driven cold inducible RNA binding protein phase separation

Intrinsically disordered proteins and proteins containing intrinsically disordered regions are highly abundant in the proteome of eukaryotes and are extensively involved in essential biological functions. More recently, their role in the organization of biomolecular condensates has become evident and along with their misregulation in several neurologic disorders. Currently, most studies involving these proteins are carried out in vitro and using purified proteins. Given that in cells, condensate‐forming proteins are exposed to high, millimolar concentrations of cellular metabolites, we aimed to reveal the interactions of cellular metabolites and a representative condensate‐forming protein. Here, using the arginine–glycine/arginine–glycine–glycine (RG/RGG)‐rich cold inducible RNA binding protein (CIRBP) as paradigm, we studied binding of the cellular metabolome to CIRBP. We found that most of the highly abundant cellular metabolites, except nucleotides, do not directly bind to CIRBP. ATP, ADP, and AMP as well as NAD⁺, NADH, NADP⁺, and NADPH directly interact with CIRBP, involving both the folded RNA‐recognition motif and the disordered RG/RGG region. ATP binding inhibited RNA‐driven phase separation of CIRBP. Thus, it might be beneficial to include cellular metabolites in in vitro liquid–liquid phase separation studies of RG/RGG and other condensate‐forming proteins in order to better mimic the cellular environment in the future.     

Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression

Intron removal from a pre-mRNA by RNA splicing was once thought to be controlled mainly by intron splicing signals. However, viral and other eukaryotic RNA exon sequences have recently been found to regulate RNA splicing, polyadenylation, export, and nonsense-mediated RNA decay in addition to their coding function. Regulation of alternative RNA splicing by exon sequences is largely attributable to the presence of two majorcis-acting elements in the regulated exons, the exonic splicing enhancer (ESE) and the suppressor or silencer (ESS). Two types of ESEs have been verified from more than 50 genes or exons: purine-rich ESEs, which are the more common, and non-purine-rich ESEs. In contrast, the sequences of ESSs identified in approximately 20 genes or exons are highly diverse and show little similarity to each other. Through interactions with cellular splicing factors, an ESE or ESS determines whether or not a regulated splice site, usually an upstream 3 splice site, will be used for RNA splicing. However, how these elements function precisely in selecting a regulated splice site is only partially understood. The balance between positive and negative regulation of splice site selection likely depends on thecis-element's identity and changes in cellular splicing factors under physiological or pathological conditions.     

SARS‐CoV‐2 nucleocapsid protein phase‐separates with RNA and with human hnRNPs

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and assemble within viral factories, dynamic compartments formed within the host cells associated with human stress granules. Here, we test the possibility that the multivalent RNA‐binding nucleocapsid protein (N) from severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) condenses with RNA via liquid–liquid phase separation (LLPS) and that N protein can be recruited in phase‐separated forms of human RNA‐binding proteins associated with SG formation. Robust LLPS with RNA requires two intrinsically disordered regions (IDRs), the N‐terminal IDR and central‐linker IDR, as well as the folded C‐terminal oligomerization domain, while the folded N‐terminal domain and the C‐terminal IDR are not required. N protein phase separation is induced by addition of non‐specific RNA. In addition, N partitions in vitro into phase‐separated forms of full‐length human hnRNPs (TDP‐43, FUS, hnRNPA2) and their low‐complexity domains (LCs). These results provide a potential mechanism for the role of N in SARS‐CoV‐2 viral genome packing and in host‐protein co‐opting necessary for viral replication and infectivity.     

Regulation of alternative splicing of Bcl-x by IL-6, GM-CSF and TPA

The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of Bcl-x pre-mRNA splicing by extracellular factors and their distinct requirements for pre-mRNA elements. In K562 leukemia cells, treatment with interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF) reduced the proportion of the Bcl-xL variant mRNA while treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) had no effect. In U251 glioma cells, however, TPA efficiently increased the Bcl-xL level. These regulations were also seen for a transfected splicing reporter mini-gene. Further analyses of deletion mutants indicate that nucleotides 1-176 of the downstream intron are required for the IL-6 effect, whereas additional nucleotides 177-284 are essential for the GM-CSF effect. As for the TPA effect, only nucleotides 1-76 are required in the downstream intron. Thus, IL-6, GM-CSF and TPA differentially regulate Bcl-x splicing and require specific intronic pre-mRNA sequences for their respective effects.