Interpretation, Stratification and Evidence for Sequence Variants Affecting mRNA Splicing in Complete Human Genome Sequences

Information theory-based methods have been shown to be sensitive and specific for pre- dicting and quantifying the effects of non-coding mutations in Mendelian diseases. We present the Shannon pipeline software for genome-scale mutation analysis and provide evidence that the soft- ware predicts variants affecting mRNA splicing. Individual information contents （in bits） of refer- ence and variant splice sites are compared and significant differences are annotated and prioritized. The software has been implemented for CLC-Bio Genomics platform. Annotation indicates the context of novel mutations as well as common and rare SNPs with splicing effects. Potential natural and cryptic mRNA splicing variants are identified, and null mutations are distinguished from leaky mutations. Mutations and rare SNPs were predicted in genomes of three cancer cell lines （U2OS, U251 and A431）, which were supported by expression analyses. After filtering, tractable numbers of potentially deleterious variants are predicted by the software, suitable for further laboratory investigation. In these cell lines, novel functional variants comprised 6-17 inactivating mutations, 1 5 leaky mutations and 6-13 cryptic splicing mutations. Predicted effects were validated by RNA-seq analysis of the three aforementioned cancer cell lines, and expression microarray analysis of SNPs in HapMap cell lines.

CD8+T cell metabolic flexibility elicited by CD28-ARS2 axisdriven alternative splicing of PKM supports antitumor immunity

Metabolic flexibility has emerged as a critical determinant of CD8+T-cell antitumor activity,yet the mechanisms driving the metabolic flexibility of T cells have not been determined.In this study,we investigated the influence of the nuclear cap-binding complex(CBC)adaptor protein ARS2 on mature T cells.In doing so,we discovered a novel signaling axis that endows activated CD8+T cells with flexibility of glucose catabolism.ARS2 upregulation driven by CD28 signaling reinforced splicing factor recruitment to pre-mRNAs and affected approximately one-third of T-cell activation-induced alternative splicing events.Among these effects,the CD28-ARS2 axis suppressed the expression of the M1 isoform of pyruvate kinase in favor of PKM2,a key determinant of CD8+T-cell glucose utilization,interferon gamma production,and antitumor effector function.Importantly,PKM alternative splicing occurred independently of CD28-driven PI3K pathway activation,revealing a novel means by which costimulation reprograms glucose metabolism in CD8+T cells.

Conservation of IRE1-Regulated bZIP74 mRNA Unconventional Splicing in Rice （Oryza sativa L.） Involved in ER Stress Responses

Protein folding in the endoplasmic reticulum （ER） is a fundamental process in plant cells that is vulnerable to many environmental stresses. When unfolded or misfolded proteins accumulate in the ER, the well-conserved unfolded protein response （UPR） is initiated to mitigate the ER stress by enhancing the protein folding capability and/or accelerating the ER-associated protein degradation. Here, we report the conservation of the activation mechanism of OsbZIP74 （also known as OsbZIP50）, an important ER stress regulator in monocot plant rice （Oryza sativa L.）. Under normal conditions, OsbZIP74 mRNA encodes a basic leucine-zipper transcription factor with a putative transmembrane domain. When treating with ER stress-inducing agents such as tunicamycin and DTT, the conserved double stem-loop structures of OsbZIP74 mRNA are spliced out. Thereafter, the resulting new OsbZIP74 mRNA produces the nucleus-localized form of OsbZIP74 protein, eliminating the hydrophobic region. The activated form of OsbZIP74 has transcriptional activation activity in both yeast cells and Arabidopsis leaf protoplasts. The induction of OsbZIP74 splicing is much suppressed in the OsIRE1 knock- down rice plants, indicating the involvement of OslRE1 in OsbZIP74 splicing. We also demonstrate that the unconventional splicing of OsbZIP74 mRNA is associated with heat stress and salicylic acid, which is an important plant hormone in systemic acquired resistance against pathogen or parasite.

The enigmatic ERH protein:its role in cell cycle,RNA splicing and cancer

Enhancer of rudimentary homolog(ERH)is a small,highly conserved protein among eukaryotes.Since its discovery nearly 20 years ago,its molecular function has remained enigmatic.It has been implicated to play a role in transcriptional regulation and in cell cycle.We recently showed that ERH binds to the Sm complex and is required for the mRNA splicing of the mitotic motor protein CENP-E.Fur-thermore,cancer cells driven by mutations in the KRAS oncogene are particularly sensitive to RNAi-mediated suppression of ERH function,and ERH expression is inversely correlated with survival in colorectal cancer pa-tients whose tumors harbor KRAS mutation.These recent fi ndings indicate that ERH plays an important role in cell cycle through its mRNA splicing activity and is critically required for genomic stability and cancer cell survival.