The Role of LUC7L2 in Splicing and MDS
Authors: Hershberger Courtney Elizabeth , Hosono Naoko , Singh Jarnail , Dietrich Rosemary C , Gu Xiaorong , Makishima Hideki , Saunthararajah Yogen , Maciejewski Jaroslaw P , Padgett Richard A 
Published: October 2019

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.