SUMMARY Mutations in SF3B1, which encodes a spliceosome component, are associated with poor outcome in chronic lymphocytic leukemia (CLL), but how these contribute to CLL progression remains poorly understood. We undertook a transcriptomic characterization of primary human CLL cells to identify transcripts and pathways affected by SF3B1 mutation. Splicing alterations, identified in the analysis of bulk cells, were confirmed in single SF3B1-mutated CLL cells and also found in cell lines ectopically expressing mutant SF3B1. SF3B1 mutation was found to dysregulate multiple cellular functions including DNA damage response, telomere maintenance, and Notch-signaling—mediated through KLF8 upregulation, increased TERC and TERT expression, or altered splicing of DVL2 transcript, respectively. SF3B1 mutation appears to be a mechanism by which changes in diverse cancer-related pathways are generated.

Summary Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in ALS/FTD patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing but not other types of RNA processing. Proteomic and biochemical analyses identified U2 snRNP as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm in C9ORF72 patient-iPSC derived motor neurons and also in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2 snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ~44% of the mis-spliced cassette exons in C9ORF72 patient brains.

We previously showed that mRNAs synthesized from three genes that naturally lack introns contain a portion of their coding sequence, known as a cytoplasmic accumulation region (CAR), which is essential for stable accumulation of the intronless mRNAs in the cytoplasm. The CAR in each mRNA is unexpectedly large, ranging in size from ∼160 to 285 nt. Here, we identified one or more copies of a 10-nt consensus sequence in each CAR. To determine whether this element (designated CAR-E) functions in cytoplasmic accumulation of intronless mRNA, we multimerized the most conserved CAR-E and inserted it upstream of β-globin cDNA, which is normally retained/degraded in the nucleus. Significantly, the tandem CAR-E, but not its antisense counterpart, rescued cytoplasmic accumulation of β-globin cDNA transcripts. Moreover, dinucleotide mutations in the CAR-E abolished this rescue. We show that the CAR-E, but not the mutant CAR-E, associates with components of the TREX mRNA export machinery, the Prp19 complex and U2AF2. Moreover, knockdown of these factors results in nuclear retention of the intronless mRNAs. Together, these data suggest that the CAR-E promotes export of intronless mRNA by sequence-dependent recruitment of the mRNA export machinery.

Post-transcriptional modifications of transfer RNAs (tRNAs) have long been recognized to play crucial roles in regulating the rate and fidelity of translation. However, the extent to which they determine global protein production remains poorly understood. Here we use quantitative proteomics to show a direct link between wobble uridine 5-methoxycarbonylmethyl (mcm5) and 5-methoxy-carbonyl-methyl-2-thio (mcm5s2) modifications catalyzed by tRNA methyltransferase 9 (Trm9) in tRNAArg(UCU) and tRNAGlu(UUC) and selective translation of proteins from genes enriched with their cognate codons. Controlling for bias in protein expression and alternations in mRNA expression, we find that loss of Trm9 selectively impairs expression of proteins from genes enriched with AGA and GAA codons under both normal and stress conditions. Moreover, we show that AGA and GAA codons occur with high frequency in clusters along the transcripts, which may play a role in modulating translation. Consistent with these results, proteins subject to enhanced ribosome pausing in yeast lacking mcm5U and mcm5s2U are more likely to be down-regulated and contain a larger number of AGA/GAA clusters. Together, these results suggest that Trm9-catalyzed tRNA modifications play a significant role in regulating protein expression within the cell.

The TREX complex (TREX) plays key roles in nuclear export of mRNAs. However, little is known about its transcriptome-wide binding targets. We used individual cross-linking and immunoprecipitation (iCLIP) to identify the binding sites of ALYREF, an mRNA export adaptor in TREX, in human cells. Consistent with previous in vitro studies, ALYREF binds to a region near the 5′ end of the mRNA in a CBP80-dependent manner. Unexpectedly, we identified PABPN1-dependent ALYREF binding near the 3′ end of the mRNA. Furthermore, the 3′ processing factor CstF64 directly interacts with ALYREF and is required for the overall binding of ALYREF on the mRNA. In addition, we found that numerous middle exons harbor ALYREF binding sites and identified ALYREF-binding motifs that promote nuclear export of intronless mRNAs. Together, our study defines enrichment of ALYREF binding sites at the 5′ and the 3′ regions of the mRNA in vivo, identifies export-promoting ALYREF-binding motifs, and reveals CstF64- and PABPN1-mediated coupling of mRNA nuclear export to 3′ processing.

SUMMARY SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its role in the pathogenesis of CLL remain elusive. Here, we show that conditional expression of Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters cell development, and induces a state of cellular senescence. Combination with Atm deletion leads to the overcoming of cellular senescence and the development of CLL-like disease in elderly mice. These CLL-like cells show genome instability and dysregulation of multiple CLL-associated cellular processes, including deregulated B cell receptor (BCR) signaling, which we also identified in human CLL cases. Notably, human CLLs harboring SF3B1 mutations exhibit altered response to BTK inhibition. Our murine model of CLL thus provides insights into human CLL disease mechanisms and treatment.

Mutations in FUS cause amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to neurodegeneration remain obscure. We previously found that U1 snRNP is the most abundant FUS interactor. Here, we report that components of the U1 snRNP core particle (Sm proteins and U1 snRNA), but not the mature U1 snRNP-specific proteins (U1-70K, U1A and U1C), co-mislocalize with FUS to the cytoplasm in ALS patient fibroblasts harboring mutations in the FUS nuclear localization signal (NLS). Similar results were obtained in HeLa cells expressing the ALS-causing FUS R495X NLS mutation, and mislocalization of Sm proteins is RRM-dependent. Moreover, as observed with FUS, knockdown of any of the U1 snRNP-specific proteins results in a dramatic loss of SMN-containing Gems. Significantly, knockdown of U1 snRNP in zebrafish results in motor axon truncations, a phenotype also observed with FUS, SMN and TDP-43 knockdowns. Our observations linking U1 snRNP to ALS patient cells with FUS mutations, SMN-containing Gems, and motor neurons indicate that U1 snRNP is a component of a molecular pathway associated with motor neuron disease. Linking an essential canonical splicing factor (U1 snRNP) to this pathway provides strong new evidence that splicing defects may be involved in pathogenesis and that this pathway is a potential therapeutic target.

The aim of our study was to investigate the protective effects and underlying mechanisms of myricetin, a bioactive food compound, on brain injury and neurological deficits after ischemic stroke. Treatment of myricetin significantly attenuated oxygen-glucose deprivation (OGD)-induced cell death in SHSY5Y cells in vitro. In a rat model of cerebral ischemia, myricetin was administered intragastrically at 2 h before and every day after middle cerebral artery occlusion (MCAO). The effects of myricetin were evaluated by various biochemical assays and neurobehavioral tests. Treatment with myricetin resulted in decreased infarction volume, reduced neuronal loss as well as lessened production of reactive oxygen species (ROS) and malondialdehyde following MCAO. We also found evidence that myricetin treatment could enhance the activity of antioxidant enzymes and mitochondrial function. Meanwhile, myricetin treatment reversed the suppression of Nrf2 nuclear translocation, and increased HO-1 expression in the ipsilateral ischemic brain and in the normal brain. Moreover, our results suggested that myricetin treatment resulted in significant improvement in neurological function. In conclusion, treatment with myricetin attenuates brain injury and neurological deficits in a rat model of cerebral ischemia via improvement of mitochondrial function and activation of the Nrf2 pathway.