hnRNP F influences binding of a 64-kilodalton subunit of cleavage stimulation factor to mRNA precursors in mouse B cells - PubMed (original) (raw)
hnRNP F influences binding of a 64-kilodalton subunit of cleavage stimulation factor to mRNA precursors in mouse B cells
K L Veraldi et al. Mol Cell Biol. 2001 Feb.
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 heavy-chain 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.
Figures
FIG. 1
IgM transcription unit and substrates used for in vitro studies. (A) Schematic representation of the Ig μ heavy-chain transcription unit. Boxes, exons; lines, introns; boxes with diagonal lines, secretory-form-specific coding sequence; boxes with horizontal lines, membrane-specific exons; angled line, potential splicing of the CH4 exon to the M1 exon. (B and C) Ig μ secretory-form-specific constructs (B) and Ig μ membrane-specific constructs (C) used for in vitro studies. The mu-sec construct contains the wild-type Ig μ heavy-chain sec-pA site and surrounding elements in a pGEM vector. The mu-sec(U→G) mutations disrupt the upstream AU-rich region in four sites, while in mu-sec(ΔGU) the downstream GU-rich element is deleted. Arrows, cleavage sites for poly(A) addition. The mu-mb construct contains the wild-type Ig mu-mb-pA site and surrounding elements. The mu-mb(U→G) substrate has-a mutation in the single mu-mb AAUAAA hexanucleotide poly(A) signal. nts, nucleotides.
FIG. 2
CstF-64 cross-linking differs while the amount of protein remains the same in different B-cell stages. (A) UV cross-linking pattern. The A20 (memory or lymphoma) and AxJ (plasma or myeloma) nuclear extracts were incubated with the indicated 32P-labeled constructs under conditions permitting polyadenylation complex formation. The proteins were UV cross-linked to the RNAs, immunoprecipitated with the polyclonal chicken anti-CstF-64 antibody, and separated by SDS–8% PAGE as described in Materials and Methods. (B) Western blot of CstF-64 showing equal amounts in the two extracts. Proteins from the indicated nuclear extracts (5 μg) were separated by SDS–10% PAGE and electroblotted to PVDF. The membrane was probed with either the polyclonal chicken antibody to recombinant CstF-64 (lanes 1 and 2) or the rabbit antibody raised to the COOH terminus of human CstF-64 (lanes 3 and 4). As a loading control, the blot was then probed with an antibody against PTB.
FIG. 3
The hnRNP F-to-H or -H′ ratio is reduced in plasma cells. The indicated whole-cell lysates (5 μg per lane) were separated by SDS–10% PAGE and electroblotted to PVDF, and the blot was probed with a mixture of antibodies recognizing hnRNP H and H′ and hnRNP F as described in Materials and Methods. As a loading control, the blot was then probed with an antibody against GAPDH. The cell lines in lanes 1 to 5 represent various stages of B-cell development: lane 1, pre-B; lane 2, early B; lane 3, memory B; lane 4, a fusion of memory and plasma cells with the phenotype of a plasma cell; lane 5, plasma cell. HeLa cell lysate (5 μg) was run in lane 6 as a size marker.
FIG. 4
Large complexes containing hnRNPs H and H′ and F form on the weak Ig mu-sec-pA site in memory B-cell but not plasma cell nuclear extracts. Biotinylated and 32P-labeled mu-sec pre-mRNA was incubated with A20 memory B-cell or AxJ plasma cell nuclear extract under conditions permitting poly(A) complex formation. The reaction mixture was fractionated by gel filtration on Sephacryl S500 as described in Materials and Methods. (A) Elution profiles of mu-sec RNA from the column. The fractions pooled for subsequent analysis are indicated. The radioactivity in 10% of each fraction was determined for the RNA profile. Column void and total included volumes (Vo and Vtot, respectively) and the elution volume of protein size marker thyroglobulin (M r = 669,000), whose profile was monitored by measuring optical density at 280 nm in a parallel column, are also shown. Lines below the x axis, fractions pooled for subsequent analyses. (B) Column elution profile before affinity selection of complexes. An aliquot representing 10% of the total proteins from each indicated pool was removed prior to the affinity selection step and analyzed by SDS–8% PAGE and Western immunoblotting with the indicated antibodies. (C) Column elution profile of affinity-purified proteins. The indicated fractions were pooled, and the proteins bound to the biotinylated RNA in each were affinity purified by avidin-agarose and eluted by RNAse A and SDS treatment. Proteins from each pool were analyzed by SDS–8% PAGE and Western immunoblotting with the indicated antibodies. Lanes NE, 2 μg of nuclear extract; lanes nb, nonbiotinylated RNA incubated with 300 μg of nuclear extract and run through the affinity purification steps as a control for background binding to the affinity resin.
FIG. 5
The region downstream of the poly(A) site facilitates binding of hnRNP H, H′, and F to the Ig mu-sec-pA site. The indicated biotinylated and 32p-labeled pre-mRNAs were incubated with A20 (memory B-cell) nuclear extracts under conditions permitting poly(A) complex formation. The reaction mixtures were fractionated by gel filtration and affinity purified as described for Fig. 4C and in Materials and Methods. Affinity-purified proteins from pooled fractions were analyzed by SDS–8% PAGE and Western immunoblotted with the indicated antibodies. (A) Proteins bound to mu-sec pre-mRNA with multiple U-to-G mutations in the AU-rich poly(A) signal region. (B) Proteins bound to mu-sec pre-mRNA truncated to remove the GU-rich downstream region. NE, nuclear extract; nb, no-biotin control. NE and nonbiotinylated RNA were prepared as described for Fig. 4.
FIG. 6
Large complexes containing hnRNPs H, H′, and F form on the Ig mu-mb-pA site in memory B-cell but not plasma cell extracts. Biotinylated and 32p-labeled mu-mb or mu-mb(U→G) mutated pre-mRNA was incubated with A20 memory B-cell or AxJ plasma cell nuclear extract under poly(A) complex-forming conditions, and the reaction mixture was fractionated by gel filtration and affinity purified as described in Materials and Methods. Both affinity-purified fractions (affinity) and 10% of the column fractions prior to affinity selection (unselected) from each pool were analyzed by SDS–8% PAGE and Western immunoblotting with an antibody against hnRNP H and H′ and then hnRNP F in two steps. The position of the affinity-purified CstF-64 in the samples (A and C) was identical to that seen in Fig. 4C. (A) mu-mb pre-mRNA construct with memory (A20) nuclear extract. (B) mu-mb pre-mRNA site with U-to-G mutation incubated with memory B-cell (A20) nuclear extract. (C) mu-mb pre-mRNA site with plasma cell (AxJ) nuclear extract.
FIG. 7
Recombinant hnRNP F binding to pre-mRNA is influenced by the region downstream of the poly(A) site and by hnRNP H′. (A) RNA substrates containing SVL pre-mRNA or SVL pre-mRNA with nearly the entire region downstream of the CstF binding site replaced with polylinker sequences (SVL-Gem) were incubated with 200 ng of recombinant hnRNP F (lanes +), and protein-RNA complexes were analyzed on a nondenaturing 5% acrylamide gel. Lanes −, input RNA only. (B) RNA substrate containing SVL pre-mRNA (lane 1) was incubated with 200 ng of recombinant hnRNP F (lane 2) and increasing concentrations of hnRNP H′ (lane 3, 10 ng; lane 4, 12 ng; lane 5, 15 ng; lane 6, 17 ng; lane 7, 20 ng; lane 8, 22 ng; lane 9, 25 ng; lane 10, 30 ng). Complexes were analyzed for panel A. Arrows, migration of the markers: bottom arrow, RNA probe alone; middle arrow (F), hnRNP F plus probe; top arrow (H), hnRNP H′ plus probe.
FIG. 8
Alteration of the hnRNP H′-to-F ratio influences CstF-64 RNA-binding activity. Recombinant hnRNP F or hnRNP H′ proteins were added to either 32P-labeled mu-sec or mu-mb pre-RNA prior to the addition of the AxJ nuclear extract. Reaction mixtures were incubated at 30°C for 5 min, placed on ice, and irradiated with UV light as described in Materials and Methods. Unprotected RNA was digested with RNase A, the proteins were immunoprecipitated with anti-CstF-64 antibody, and the 32p-tagged CstF-64 was electrophoresed on SDS–7.5% PAGE gel and quantified by densitometry. An anti-COOH-terminal CstF-64 antibody was used for the immunoprecipitations. Lanes 1 and 6, no recombinant protein; lanes 2 to 4, recombinant hnRNP F (0.25, 0.5, and 1 μg respectively), added; lane 5, 1 μg of recombinant H′ added.
FIG. 9
Recombinant hnRNP F inhibits the nuclear cleavage reaction. The 32p-labeled SVL pre-mRNA was incubated in the in vitro cleavage system from plasma cells for 30 min at 30°C. Products were analyzed on a 5% acrylamide gel containing 8 M urea. The positions of the input pre-mRNA and the 5′ cleavage product are indicated. Lanes 1 to 7 and 9 to 11 contain nuclear extract, while in lanes 8 and 12 no nuclear extract was added. Lane 1, no recombinant protein; lane 2, 100 ng of recombinant H′ (rH′); lane 3, 300 ng of rH′; lane 4, 600 ng of rH′; lane 5, 100 ng of recombinant F (rF); lane 6, 300 ng of rF; lane 7, 600 ng of rF; lane 9, 100 ng of rF plus 500 ng of rH′; lane 10, 300 ng of rF plus 300 ng of rH′; lane 11, 500 ng of rF plus 100 ng of rH′; lane 13, 32P-labeled runoff transcript of an SVL template cut with _Hin_cII that is 6 nucleotides shorter than the cleavage product. Recombinant hnRNP F and H′ proteins were purified as described in Materials and Methods; recombinant proteins were preincubated with the RNA for 5 min at room temperature prior to the addition of the nuclear extract.
FIG. 10
Western analysis of the hnRNP F transfectants. Proteins from AxJ plasma cells or transfectants of AxJ with hnRNP F linked to the Flag-tagged epitope were separated on an SDS–10% PAGE gel and blotted to membranes. The positions of the authentic hnRNP F and the Flag-tagged F protein are indicated. (A) One gel was run and cut in half vertically. Lanes 1 and 2 were probed with antibodies to the Flag epitope, while lanes 3 and 4 were probed with antibodies to hnRNP F. Lanes 1 and 4 contain AxJ cell lysates, while lanes 2 and 3 contain lysate from AxJ transfectant B4. (B) Nine individual G418-resistant clones were isolated and analyzed. Asterisks indicate transformants chosen for further study. Lane 1, transformant B6; lane 2, B5; lane 3, B4; lane 4, C4; lane 5, C5; lane 6, C6; lane 7, D4; lane 8, D5; lane 9, D6.
FIG. 11
Northern analysis of the hnRNP F transfectants. Poly(A)+ RNA was isolated from AxJ cells or the transfected AxJ cells, run on an formamide-formaldehyde-containing 0.8% agarose gel, blotted, and then probed sequentially with 32P-labeled antisense RNA derived from the IgG2a CH3 exon and then GAPDH. The locations of the 1.8- and 3.6-kb messages bearing, respectively, the sec-pA site (sec) and the mb-pA site (mb) are indicated. The amount of each Ig heavy-chain species and the amount of total Ig mRNA relative to that of GAPDH were quantified in the phosphorimager analysis of four separate Northern blots. The mean values (± the standard errors) are beneath the corresponding lanes of the blot. Lane 1, values obtained with AxJ cells transfected with the empty Flag vector alone; lanes 2 to 5, values obtained from four separate Northern blots of the transfectants receiving the hnRNP F-Flag recombinant, transfectants A5, B5, D4, and D6, respectively; lane 6, RNA from untransfected AxJ. Asterisks indicate results that were considered to be extremely statistically different (P < 0.0002) from the other values by sequential, two-tailed, unpaired t tests.
FIG. 12
Model for the regulation of poly(A) site choice during B-cell development. In mature and memory B cells, hnRNP F protein levels are high while CstF-64 binding activity is low. The binding of hnRNP F and hnRNP H and H′ to the sec-pA site near the downstream GU- or U-rich element immediately after that portion of the RNA is synthesized may inhibit the association of CstF-64 with pre-mRNA and hinder the assembly of a stable polyadenylation complex at that site. Consequently, there is an increased chance that the strong downstream mb-pA site will be transcribed by a polymerase molecule still carrying the polyadenylation factors and serve as a cleavage or polyadenylation signal. Upon lymphokine stimulation and differentiation to an antibody-secreting plasma cell, hnRNP F protein expression is decreased, which leads to an increase in the apparent affinity of CstF-64 for the sec-pA site as it is transcribed. The cleavage-polyadenylation complex can form at the sec-pA site, even before the polymerase reaches the membrane site, thus favoring use of the promoter-proximal (sec) site. This plasma cell phenotype can be reversed by overexpression of recombinant hnRNP F.
References
- Beyer K, Dandekar T, Keller W. RNA ligands selected by cleavage stimulation factor contain distinct sequence motifs that function as downstream elements in 3′-end processing of pre-mRNA. J Biol Chem. 1997;272:26769–26779. - PubMed
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