During nuclear maturation of most eukaryotic pre-messenger RNAs and long non-coding RNAs, introns are removed through the process of RNA splicing. Different classes of introns are excised by the U2-type or the U12-type spliceosomes, large complexes of small nuclear ribonucleoprotein particles and associated proteins. We created intronIC, a program for assigning intron class to all introns in a given genome, and used it on 24 eukaryotic genomes to create the Intron Annotation and Orthology Database (IAOD). We then used the data in the IAOD to revisit several hypotheses concerning the evolution of the two classes of spliceosomal introns, finding support for the class conversion model explaining the low abundance of U12-type introns in modern genomes.

Myeloid neoplasms are characterized by frequent mutations in at least seven components of the spliceosome that have distinct roles in the process of pre-mRNA splicing. Hotspot mutations in SF3B1 , SRSF2 , U2AF1 and loss of function mutations in ZRSR2 have revealed widely different aberrant splicing signatures with little overlap. However, previous studies lacked the power necessary to identify commonly mis-spliced transcripts in heterogeneous patient cohorts. By performing RNA-Seq on bone marrow samples from 1,258 myeloid neoplasm patients and 63 healthy bone marrow donors, we identified transcripts frequently mis-spliced by mutated splicing factors (SF), rare SF mutations with common alternative splicing (AS) signatures, and SF-dependent neojunctions. We characterized 17,300 dysregulated AS events using a pipeline designed to predict the impact of mis-splicing on protein function. Meta-splicing analysis revealed a pattern of reduced levels of retained introns among disease samples that was exacerbated in patients with splicing factor mutations. These introns share characteristics with “detained introns,” a class of introns that have been shown to promote differentiation by detaining pro-proliferative transcripts in the nucleus. In this study, we have functionally characterized 17,300 targets of mis-splicing by the SF mutations, identifying a common pathway by which AS may promote maintenance of a proliferative state.

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Highlights d LUC7-like proteins are U1 snRNP components and alternative splicing regulators d LUC7L2 and LUC7L3 share an evolutionarily conserved role in 5 0 splice site selection d LUC7-like proteins bind distinct factors regulating unique alternative splicing events d Loss of LUC7L2 alters spliceosome and glycolytic genes that may contribute to disease

Decrease in DNA dioxygease activity generated by TET2 gene family is crucial in myelodysplastic syndromes (MDS). The general down-regulation of 5-hydroxymethylcytosine (5-hmC) argues for a role of DNA demethylation in MDS beyond TET2 mutations, which albeit frequent, do not convey any prognostic significance. We investigated TETs expression to identify factors which can modulate the impact of mutations and thus 5-hmC levels on clinical phenotypes and prognosis of MDS patients. DNA/RNA-sequencing and 5-hmC data were collected from 1,665 patients with MDS and 91 controls. Irrespective of mutations, a significant fraction of MDS patients exhibited lower TET2 expression, while 5-hmC levels were not uniformly decreased. In searching for factors explaining compensatory mechanisms, we discovered that TET3 was up-regulated in MDS and inversely correlated with TET2 expression in wild-type cases. While TET2 was reduced across all age-groups, TET3 levels were increased in a likely feedback mechanism induced by TET2 dysfunction. This inverse relationship of TET2 and TET3 expression also corresponded to the expression of L-2-hydroxyglutarate dehydrogenase, involved in agonist/antagonist substrate metabolism. Importantly, elevated TET3 levels influenced the clinical phenotype of TET2-deficiency whereby the lack of compensation by TET3 (low TET3 expression) was associated with poor outcomes of TET2 mutant carriers.

Background Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide. Improved tools are needed for detecting HCC so that treatment can begin as early as possible. Current diagnostic approaches and existing biomarkers, such as alpha‐fetoprotein (AFP) lack sensitivity, resulting in too many false negative diagnoses. Machine learning may be able to identify combinations of biomarkers that provide more robust predictions and improve sensitivity for detecting HCC. We sought to evaluate whether metabolites in patient saliva could distinguish those with HCC, cirrhosis, and those with no documented liver disease. Methods and Results We tested 125 salivary metabolites from 110 individuals (43 healthy, 37 HCC, 30 cirrhosis) and identified four metabolites that displayed significantly different abundance between groups (FDR P < .2). We also developed four tree‐based, machine‐learning models, optimized to include different numbers of metabolites, that were trained using cross‐validation on 99 patients and validated on a withheld test set of 11 patients. A model using 12 metabolites –octadecanol, acetophenone, lauric acid, 1‐monopalmitin, dodecanol, salicylaldehyde, glycyl‐proline, 1‐monostearin, creatinine, glutamine, serine and 4‐hydroxybutyric acid – had a cross‐validated sensitivity of 84.8%, specificity of 92.4% and correctly classified 90% of the HCC patients in the test cohort. This model outperformed previously reported sensitivities and specificities for AFP (20‐100 ng/mL) (61%, 86%) and AFP plus ultrasound (62%, 88%). Conclusions and Impact Metabolites detectable in saliva may represent products of disease pathology or a breakdown in liver function. Notably, combinations of salivary metabolites derived from machine learning may serve as promising non‐invasive biomarkers for the detection of HCC.

Additional sex combs-like 1 (ASXL1), an epigenetic modulator, is frequently mutated in myeloid neoplasms. Recent analyses of mutant ASXL1 conditional knock-in (ASXL1-MT-KI) mice suggested that ASXL1-MT alone is insufficient for myeloid transformation. In our previous study, we utilized retrovirus-mediated insertional mutagenesis, which exhibited susceptibility of ASXL1-MT-KI hematopoietic cells to transform into myeloid leukemia cells. In this screening, we identified Hematopoietically expressed homeobox (HHEX) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. Expression of HHEX enhanced proliferation of ASXL1-MT expressing HSPCs by inhibiting apoptosis and blocking differentiation, whereas it showed only modest effect in normal HSPCs. Moreover, ASXL1-MT and HHEX accelerated the development of RUNX1-ETO9a and FLT3-ITD leukemia. Conversely, HHEX depletion profoundly attenuated the colony-forming activity and leukemogenicity of ASXL1-MT-expressing leukemia cells. Mechanistically, we identified MYB and ETV5 as downstream targets for ASXL1-MT and HHEX by using transcriptome and ChIP-seq analyses. Moreover, we found that expression of ASXL1-MT enhanced the binding of HHEX to the promoter loci of MYB or ETV5 via reducing H2AK119ub. Depletion of MYB or ETV5 induced apoptosis or differentiation in ASXL1-MT-expressing leukemia cells, respectively. In addition, ectopic expression of MYB or ETV5 reversed the reduced colony-forming activity of HHEX-depleted ASXL1-MT-expressing leukemia cells. These findings indicated that the HHEX-MYB/ETV5 axis promotes myeloid transformation in ASXL1-mutated preleukemia cells.

Chromosomal abnormalities are common in myelodysplastic syndrome (MDS) and other myeloid malignancies. -7/del7q is found in 15% of MDS patients, but the pathological consequences of this deletion are unknown. To identify genes in the -7/del7q region that may contribute to pathogenesis of MDS, whole exome sequencing of MDS patient samples was used to detect novel somatic nonsense/frameshift mutations. Novel somatic mutations in the gene LUC7L2 were found in 27 malignant cases. Cases of mutant LUC7L2 hemizygosity, heterozygosity and homozygosity were observed in this cohort. The mutations were frameshift or nonsense, resulting in a premature stop codon and decreased expression of the LUC7L2 mRNA. LUC7L2 is located in the most commonly deleted region of -7/del7q; 7q34, which is deleted in 85% of -7/del7q patients. Patients with LUC7L2 mutations and those harboring -7/del7q have similar and statistically shorter overall survival than those with normal LUC7L2 expression levels. LUC7L2 is a poorly characterized, splicing-related protein. The function of LUC7L2 is largely unknown, but it is the ortholog of yeast protein LUC7p, which is involved in recruitment of early splicing factors. Recent studies have shown that over 65% of MDS patients harbor mutations in one of several proteins involved in pre-mRNA splicing. Therefore, we hypothesize that LUC7L2 acts as a mammalian splicing factor and deficiency of LUC7L2 results in aberrant splicing of transcripts that contribute to the pathogenesis of MDS. We characterized the role LUC7L2 in splicing by showing that LUC7L2 interacts with splicing factors using immunoprecipitation followed by mass spectrometry. Immunoprecipitation of LUC7L2 and interacting proteins revealed that LUC7L2 interacts with many proteins in the SR and HnRNP families of splicing regulators suggesting that LUC7L2 may also play a role as a splicing regulator. Splicing regulatory proteins are a diverse group that can influence constitutive splicing as well as alternative splicing. Therefore, to understand the mRNA targets of LUC7L2, we performed RNA CLIP-Seq. This revealed that LUC7L2 binds at least 301 pre-mRNA transcripts as well as spliceosomal snRNAs. LUC7L2 binding is enriched near splice junctions, with 117 of binding sites lying within 100bp of a splice site. To understand how LUC7L2 regulates splicing, we knocked down LUC7L2 in HEK293 cells and subjected the cells to a qRT-PCR based intron splicing efficiency assay as well as a splicing sensitive microarray. The intron splicing efficiency assay was performed on a subset of LUC7L2 targets identified by CLIP-Seq and showed that LUC7L2-deficiency often promotes the splicing of introns, suggesting that LUC7L2 acts as a splicing repressor. To understand the effect of LUC7L2 deficiency on all LUC7L2-targets, the splicing sensitive microarray identified splicing changes near LUC7L2-binding sites. LUC7L2-deficiency significantly alters the splicing of 151 transcripts that contain LUC7L2 binding sites. Missplicing of transcripts can alter protein function, leading to profound repercussions in the cell. The microarray analysis of LUC7L2-deficient cells identified several misspliced transcripts that are involved in the processes of proliferation, differentiation, and apoptosis, which are disrupted in MDS hematopoietic stem cells. Exons 2, 7 and 8 of the RUNX1 transcript are frequently skipped in the LUC7L2 knockdown cells. Although the mRNA expression of RUNX1 remains the same, several downstream transcripts that are regulated by RUNX1 have significantly increased or decreased expression, suggesting altered function of the RUNX1 protein. RUNX1 is a transcription factor that is required for HSC differentiation into myeloid progenitor cells. Altered splicing of RUNX1 and changes in the RUNX1 pathway could cause defects in the HSC differentiation process. In conclusion, novel somatic mutations in have been observed in LUC7L2 in patients with MDS. LUC7L2 is a protein that acts as a splicing regulator and deficiency of this protein results in missplicing of transcripts, including factors involved in differentiation, which may contribute to the pathogenesis of MDS. Disclosures Makishima: The Yasuda Medical Foundation: Research Funding.