hnRNP F Influences Binding of a 64-Kilodalton Subunit of Cleavage Stimulation Factor to mRNA Precursors in Mouse B Cells

Abstract

Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued for trans-acting modifiers of the cleavage-polyadenylation reaction operating differentially during B-cell developmental stages. Using four complementary approaches, we demonstrate that a change in the level of hnRNP F is an important determinant in the regulated use of alternative polyadenylation sites between memory and plasma stage B cells. First, by Western analyses of cellular proteins, the ratio of hnRNP F to H or H was found to be higher in memory B cells than in plasma cells. In memory B cells the activity of CstF-64 binding to pre-mRNA, but not its amount, was reduced. Second, examination of the complexes formed on input pre-mRNA in nuclear extracts revealed large assemblages containing hnRNP H, H, and F but deficient in CstF-64 in memory B-cell extracts but not in plasma cells. Formation of these large complexes is dependent on the region downstream of the AAUAAA in pre-mRNA, suggesting that CstF-64 and the hnRNPs compete for a similar region. Third, using a recombinant protein we showed that hnRNP F could bind to the region downstream of a poly(A) site, block CstF-64 association with RNA, and inhibit the cleavage reaction. Fourth, overexpression of recombinant hnRNP F in plasma cells resulted in a decrease in the endogenous Ig heavychain mRNA secretory form-to-membrane ratio. These results demonstrate that mammalian hnRNP F can act as a negative regulator in the pre-mRNA cleavage reaction and that increased expression of F in memory B cells contributes to the suppression of the Ig heavy-chain secretory poly(A) site.The immunoglobulin (Ig) heavy-chain transcription unit and the two heavy-chain mRNAs it encodes are shown in Fig. 1A (reviewed in reference 8). In mature and memory B cells the promoter-distal membrane-specific poly(A) site (mb-pA) is selected and splicing to the downstream M1 exon occurs via a 5Ј splice site within the coding region of CH4. The secretoryspecies-specific poly(A) (sec-pA) and mb-pA sites are used with equal frequency in mature and memory B cells and their tumor analogs, lymphoma cells. Plasma cells are terminally differentiated B cells; myeloma cells are their tumor counterparts, which accurately reflect their pattern of Ig gene expression. In plasma and myeloma lines polyadenylation takes place preferentially at the weaker, promoter-proximal sec-pA site, precluding the splicing to membrane-specific exons; the sec-pA site is used up to 100-fold more often than the mb-pA site in plasma cells (23). Polyadenylation at the promoter-proximal secretory site and splicing of CH4 to M1 are mutually exclusive events; consequently, it is the balance between these two that determines the final ratio of secretory-form to membrane mRNA (sec-to-mb mRNA ratio) (26). Previous experiments demonstrated that regulation of Ig heavy-chain mRNA production occurs primarily at the level of polyadenylation, not message stability, transcription termination, or splicing efficiency (14-16, 19, 2...

TERRA transcripts are bound by a complex array of RNA-binding proteins

“…Th ese results indicate that hnRNP F may act as a potent repressor of TERRA levels. Th is repression might be linked to the known role of hnRNP F as a negative regulator of pre-mRNA cleavage, which is needed for the polyadenylation steps 27 . In particular, hnRNP F downregulation may result in increased polyA + -TERRA contributing to augmented protection of 3 ′ end of TERRA from exonucleases, which in turn could increase TERRA stability and the observed increase in TERRA levels.…”

Chimeric human CstF-77/ Drosophila Suppressor of forked proteins rescue suppressor of forked mutant lethality and mRNA 3′ end processing in Drosophila

“…In vitro, affinity of CstF for the U͞GU-rich elements, which are highly variable in sequence, defines the efficiency of poly(A) sites by determining the stability of the cleavage complex on the pre-mRNA (22). In mammalian cells, several studies have correlated shifts in the choice of poly(A) sites with quantitative or qualitative variations of CstF-64 (23)(24)(25)(26)(27). In Drosophila, we and others have shown that modulation of su(f) activity in su(f) mutants affects the utilization of alternative poly(A) sites in the f 1 mutation, the Adh͞Adhr locus, and the su(f) gene itself (19,20,28,29).…”

Hide and Go Seek: Activation of the Secretory-Specific Poly (A) Site of Igh by Transcription Elongation Factors

“…Nascent RNA cleavage in polyadenylation can be blocked by U1A (Phillips, Pachikara et al 2004;Ma, Gunderson et al 2006) or hnRNP F (Veraldi, Arhin et al 2001); the levels of both of these are high in B-cells and lower in plasma cells (summarized in Table 1) and (Ma, Gunderson et al 2006) consistent with a loss of their inhibitory function on the poly(A) sites in plasma cells. While hnRNP F might be expected to block any poly(A) site based on its sequence preference for downstream regions (Alkan, Martincic et al 2006), we found by a micro-array analyses of hnRNP F transfected cells that only some genes were affected, most notable among these were secretory Igh and ELL2, a transcription elongation factor (Martincic, Alkan et al 2009).…”

“…A shift in the SR-proteins is seen from the activating type (ASF/SF2) in B-cells to the repression type (SRp20) in plasma cells. The factors hnRNP F and U1A, both of which block the secretory-specific poly(A) site from functioning, are reduced in plasma cells (Veraldi, Arhin et al 2001;Milcarek, Martincic et al 2003;Alkan, Martincic et al 2006) and (Ma, Gunderson et al 2006) which would allow the secretory site to function better. The major effectors in the alternative processing in B versus plasma cells are summarized in Table 1.…”