Recruitment of a basal polyadenylation factor by the upstream sequence element of the human lamin B2 polyadenylation signal

Abstract

We have investigated how the upstream sequence element (USE) of the lamin B2 poly(A) signal mediates efficient 3'-end formation. In vitro analysis demonstrates that this USE increases both the efficiency of 3'-end cleavage and the processivity of poly(A) addition. Cross-linking using selectively labeled synthetic RNAs confirms that cleavage stimulation factor interacts with the sequences downstream of the cleavage site, while electrophoresis mobility shift assays demonstrate that the USE directly stabilizes the binding of the cleavage and polyadenylation specificity factor to the poly(A) signal. Thus in common with other poly(A) signals, the lamin B2 USE directly enhances the binding of basal poly(A) factors. In addition, a novel 55-kDa protein binds to the USE and the core poly(A) signal and appears to inhibit cleavage. The binding activity of this factor appears to change during the cell cycle, being greatest in S phase, when the lamin B2 gene is transcribed.

FIG. 1

Location and sequence of the lamin B2 poly(A) signal. (A) Schematic representation of the short intergenic region separating the 3′ UTR of the lamin B2 gene from the start of the downstream gene (ppv1). The open arrow indicates the lamin B2 poly(A) site, and the two filled arrows denote the two clusters of start sites mapped for ppv1 (7). The positions of transcription factor binding sites, and the location of the origin overlapping with the 3′ UTR of the lamin B2 gene, are also shown (4, 17). (B) Sequence of the wild-type 199-bp lamin B2 poly(A) signal fragment, showing the AAUAAA (underlined) and the U tracts in the USE (boldfaced). The mtRNA contains three point mutations that inactivate the AAUAAA, the USEmt RNA contains point mutations in two U tracts, and the ISEmt RNA contains changes in the sequence between the AAUAAA and the cleavage site. The other RNAs are as follows. Sp has the first 90 nt of the USE replaced by spacer sequence; USE contains the 100 nt of the USE; and the three precleaved RNAs (pre-wt, pre-mt, and pre-Sp) are identical to the full-length wt, mt, and Sp RNAs, respectively, except that they end at the normal cleavage site.

FIG. 2

The lamin B2 USE is required for efficient cleavage and poly(A) addition in vitro. (A) Cleavage assays were performed in nuclear extract using wt, Sp, USEmt, or ISEmt RNAs. The positions of the input RNAs (solid- and -open box) and the 5′ cleavage products (solid box) are indicated to the right of the gel. Note that for the Sp RNA (lanes 6 to 9), both the input and the 5′ fragment are slightly longer than those for the other RNAs. (B) Poly(A) addition reactions in nuclear extract using the pre-wt, pre-Sp, pre-mt, and D1 RNAs. [The D1 RNA has no poly(A) signal sequences.] The positions of the input and poly(A)⁺ RNAs are indicated. (C) Poly(A) addition is qualitatively different for the pre-wt and pre-Sp substrates. The graphs represent quantitation of lanes 5 and 9 of panel B—the 60-min time points. Arrows indicate the positions of the input RNA, and brackets show the extent of the heterogeneous poly(A)⁺ band.

FIG. 3

UV cross-linking of proteins to the lamin B2 poly(A) signal. (A) Cross-linking of wt, Sp, pre-wt, mt, and USE+. The migration of prestained molecular weight markers is indicated to the left. (B) Immunoprecipitation of cross-linked proteins using antibodies specific for the 64-kDa RNA-binding subunit of CstF (lane 2), hnRNP C (lane 3), and PTB (lane 4). Lane 1 shows half of the input material used in each case. Lane 6 shows precipitation of PTB cross-linked to the D1 RNA, with half of the input material shown in lane 5.

FIG. 4

Interaction of CstF and CPSF with the lamin B2 poly(A) signal. (A) Schematic of the distribution of label in the 5′* and 3′* selectively labeled RNAs and the uniformly labeled (U*) wt RNA. Solid box, AAUAAA element; arrow, cleavage site; dots, individual labeled U residues; dashes, labeled U tracts. (B) Cross-linking of the uniformly and selectively labeled RNAs in nuclear extract (Nuc. Ext.; lanes 1, 3, and 5) or purified CPSF and CstF (lanes 2, 4, and 6). (C) Stability of CPSF interacting with the pre-wt (lanes 1 to 5) and pre-Sp (lanes 6 to 10) RNAs. The CPSF-RNA complexes were separated from the free RNA following the addition of an excess of unlabeled pre-wt RNA. (D) Electrophoresis mobility shift assay using purified CPSF and pre-USEmt (lane 1), pre-Sp (lane 2), pre-mt (lane 3), and pre-wt (lane 4) RNAs. (E) Effects of DNA oligonucleotide competitors (Oligo) on the binding of CPSF to the pre-wt RNA.

FIG. 5

A negative factor binds to the lamin B2 poly(A) signal and inhibits cleavage in nuclear extract. (A) Sequence of the USE showing the locations of the five DNA oligonucleotides used as competitors in the cross-linking and cleavage reactions. (B) Cross-linking of wt RNA in nuclear extract in the presence of increasing amounts (0, 1, 10, 50, and 100 pmol) of the different DNA oligonucleotides. (C) Effects of the oligonucleotide competitors on cleavage of wt RNA. Time courses of cleavage in the absence (lanes 2 to 5) or presence (lanes 6 to 9) of 100 pmol of oligonucleotide 2 are shown, as is cleavage at 60 min in the presence of oligonucleotide 5 (lane 10). (D) Cleavage of the wt (lanes 2 and 3), Sp (lanes 4 and 5), USEmt (lanes 6 and 7), and ISEmt (lanes 8 and 9) RNAs in nuclear extract, all in the presence of 100 pmol of oligonucleotide 2.

FIG. 6

hnRNP C does not inhibit the lamin B2 poly(A) signal. (A) Western blot of 1-μl (lanes 1 and 3) and 0.2-μl (lanes 2 and 4) equivalents of mock- and hnRNP C-depleted (ΔC) nuclear extracts (NE). (B) Cross-linking of the wt RNA in untreated (UT; lane 1), mock-depleted (lane 2), or hnRNP C-depleted (ΔC; lane 3) extracts. (C) Quantitation of cleavage of the wt lamin B2 RNA and an adenovirus L3 poly(A) signal RNA in the mock-depleted and ΔC extracts. Error bars indicate standard deviations for three assays.

FIG. 7

Cell cycle-dependent changes in the proteins cross-linking to the lamin B2 poly(A) signal. (A) Nuclear run-on analysis of the lamin B2 gene using asynchronous (top) and S-phase (bottom) cells. The probes used detect RNA from histone H4 (his), 5S rRNA (5S), and the lamin B2 gene (B and ED). Probe BG controls for background hybridization. (B) Cross-linking of the wt RNA in whole-cell extracts prepared from asynchronous (A; lane 1) or synchronized (lanes 2 to 5) cells.