PP2500 mRNA, a Splice Variant of the Multiple Ankirin Repeat Single KH Domain (Mask), Is Highly Expressed in Plasma Cells of Multiple Myeloma.
Authors: Duarte Adriana S.S. , Ortega Manoela M. , Costa Fernando F. , Lima Carmen S.P. , Saad Sara T.O. 
Published: April 2020

Abstract
The Ankyrin (ANK)-repeat is one of the most common protein sequence motifs, which leads itself to variation in overall domain size by simple sequence duplication or deletion. The Mask (Multiple Ankyrin Repeats Single KH domain) gene, which codifies an ANK-repeat protein, is located in chromosome 5(q31.3) and it is composed of 39 exons. It generates isoforms by alternative 3′splicing. The first splice variant (hMask) lacks the 10A exon of the Mask gene, generating a mRNA containing 34 exons. The other, Mask-BP3ARF, results from fusion of splice variant hMask, with the two last exons of the gene Eif4Ebp3 (exons B and C) and an intermediate exon (exon 0), generating 36 exons. Recently, a new splice variant, denominated PP2500, was deposited in the data base GeneBank, it presents the first 10 exons, homologous to Mask mRNA with a poly(A+) signal and it is a new splice variant of Mask. In Drosophila, MASK protein seems to interact with members of the Receptor Tyrosine Kinase (RTK) signalling pathway and loss of this interaction increases programmed cell death, reduces cell proliferation, inhibits photoreceptor differentiation, affects RTK dependents processes but does not affect MAPK (Mitogen Activated Protein Kinase) activation. However, the biological functions of these proteins in humans remain still unknown. The aim of this study was to investigate the expression of Mask splice variants in multiple myeloma (MM). Fifteen patients with MM and 3 normal donors participated in this study. Total RNA was extracted from positively selected plasma cells in magnetic column, by Macs Microbeads antibody anti-CD138 and the percentage of purity of plasma cells varied from 78.38% to 96.02% (average 87.95%). We used as control, total RNA from positively selected plasma cells, from a culture of B normal lymphocytes of bone marrow donors (purity 88.69%). The complementary DNA (cDNA) was analyzed by Real-time detection of amplification, performed in an ABI 5700 Sequence Detector System using SybrGreen PCR Master Mix (qPCR). The b-actin gene was used as endogenous control of the reaction. The mean expression of the mRNA of the hMask and Mask-BP3ARF genes were 3 and 4 times increased, respectively, compared with control. The mean expression of the PP2500 mRNA was 14 times increased, compared with control. Quantification of hMask, Mask-BPARF and PP2500 mRNA was not influenced by age, gender, ethnic origin, stage of the disease, β2-microglobulin, serum creatinine and lactate dehydrogenase values (Fisher’s exact test, P&gt; 0.05). Previously we demonstrated that MASK is associated with SHP2, a protein tyrosine-phosphatase. In MM, SHP2 mediates the anti-apoptotic effect of Interleukin-6 (Chauhan et al. JBC275: 27845, 2000). Interleukin-6 triggers proliferation of MM cells via the MAPK cascade, which includes SHP2 activation. Thus, the increased expression of Mask splice variants in plasma cells of MM suggests that their proteins may be involved in this signaling pathway and provide an insight for novel treatment approaches in MM. Supported by FAPESP and CNPq