Transcription elongation factor ELL2 influences splicing versus poly(A) site choice in the Ig heavy chain gene

Processing of Immunoglobulin heavy chain (IgH) mRNA is a paradigm for competition between splicing and polyadenylation. In plasma cells pre-mRNA is polyadenylated mainly at the promoter-proximal secretory site while B-cells utilize a cryptic 5’ splice site in the last secretory-specific exon; these are mutually exclusive events for all IgH pre-mRNAs. Transcription elongation factor ELL2, more abundant in plasma cells relative to B-cells, was down-modulated by overexpression of heterogenous ribonucleoprotein F, a condition which reduced production of secretory IgH mRNA. Transfection of B-cells with ELL2 and the IgH reporter showed an accelerated use of the secretory poly(A) site, positioned in competition with the splice to M1; a small interfering RNA to ELL2 reduced expression of IgH secretory mRNA. Co-transcription factors ELL1 and PC4 were ineffective at driving secretory-poly(A) site use. ELL2 had little effect on poly(A) site choice with reporters containing tandem-linked poly(A) sites. Shorter forms of ELL2 protein result from both internal initiation at M186 and protein processing. An alternative splicing reporter driven by IgH or non-Ig promoters revealed that ELL2 and its M186 initiated form were able to accelerate exon skipping. Therefore, ELL2 influences IgH pre-mRNA processing through facilitating skipping of the alternative splice to the membrane form. AxJ plasma cells were stably transfected to overexpress hnRNP-F, -H or empty vector. Clones showing high overexpression levels of F or H by western blot were selected. The IgH sec to mb ratios of these clones were determined. A global gene expression analysis was performed on mRNA from two clones from hnRNP-F, which demonstrated a lower sec:mb ratio, and one from each of the controls: overexpression of hnRNP-H, or transfection with empty vector, or A20 B-cells, using Affymetrix gene micro array technology.