Improved Engraftment of Lentivirally Transduced Adipose Tissue-Derived Stem Cells for Hemophilia B Therapy Using Bioengineered Cell Sheet Technology

apoptosis. The long noncoding RNA (lncRNA) Saf was reported to participate in this process. LncRNAs are >200 nucleotide transcripts that form complexes with various RNA-binding proteins, including splicing factors, to regulate and diversify gene function. Last year, we reported that the 1.5-kb lncRNA Saf was regulated by GATA-1 and KLF1 and induced during maturation of human erythroid cells. As the precise mechanism of Saf function is unknown, we extend our fi ndings to include gene expression analysis, cellular localization, and identifi cation of Saf interacting proteins. For gene expression studies, two human cell lines, Jurkat T-cells and K562 erythroleukemia cells, were transduced with lentivirus particles encoding for Saf/ GFP or GFP alone. Total RNA was isolated from the resulting GFP+ cells and genome wide expression analyses performed on HG-U133_Plus2 cartridges. Saf over expression had no effect on global gene transcription, supporting a post-transcriptional role. To assess localization, total RNA was extracted from the nuclear and cytoplasmic fractions of MCF7 cells that endogenously express Saf. RT-PCR analysis revealed that Saf was localized in the nucleus and fraction purity verifi ed with the nuclear-retained 47S pre-rRNA. Saf associated proteins were isolated by RNA pulldown of in vitro transcribed Saf RNAs (-/+ Biotin-UTP) that were mixed with K562 nuclear lysates and recovered using anti-streptavidin beads. Mass spectrometry analysis identifi ed eight partners unique to biotinlabeled Saf. One protein with highest counts, human splicing factor (SPF45) also called RNA-binding motif protein 17 (RBM17), was confi rmed to interact with Saf by RNA co-immunoprecipation using SPF45-specifi c antibodies in nuclear lysates prepared from K562 cells with stable expression of Saf and RT-PCR. Specifi c interaction was demonstrated by the failure to detect Saf using either control IgG or GATA-1 antibodies. SPF45 regulates alternative splicing of FasR pre-mRNA by inducing exon 6 skipping to generate sFas proteins. To monitor this molecular event in maturing erythroid cells, we developed a qRT-PCR assay to indirectly measure this isoform (FasRExon6) as a ratio of total FasR mRNA levels and detected a 2.5-fold increase in these sFas-encoding transcripts in late stage adult BM-derived erythroblasts. Regulation of apoptosis is essential for erythropoiesis and many other developmental processes. Our fi nding that Saf participates in FasR alternative splicing through direct interaction with SPF45 reveals a novel layer of modulation of the cell death program that is required for the proper generation of mature red blood cells.