Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition

Abstract

At least half of all human pre-mRNAs are subject to alternative 3' processing that may modulate both the coding capacity of the message and the array of post-transcriptional regulatory elements embedded within the 3' UTR. Vertebrate poly(A) site selection appears to rely primarily on the binding of CPSF to an A(A/U)UAAA hexamer upstream of the cleavage site and CstF to a downstream GU-rich element. At least one-quarter of all human poly(A) sites, however, lack the A(A/U)UAAA motif. We report that sequence-specific RNA binding of the human 3' processing factor CFI(m) can function as a primary determinant of poly(A) site recognition in the absence of the A(A/U)UAAA motif. CFI(m) is sufficient to direct sequence-specific, A(A/U)UAAA-independent poly(A) addition in vitro through the recruitment of the CPSF subunit hFip1 and poly(A) polymerase to the RNA substrate. ChIP analysis indicates that CFI(m) is recruited to the transcription unit, along with CPSF and CstF, during the initial stages of transcription, supporting a direct role for CFI(m) in poly(A) site recognition. The recognition of three distinct sequence elements by CFI(m), CPSF, and CstF suggests that vertebrate poly(A) site definition is mechanistically more similar to that of yeast and plants than anticipated.

Figure 1.

Sequence comparison of the 3′ ends of vertebrate PAPOLA and PAPOLG genes. (A) Comparison of the sequences of the PAPOLA genes of Homo sapiens (Hs), Mus musculus (Mm), Gallus gallus (Gg), and Xenopus laevis (Xl) immediately upstream of the primary poly(A) cleavage site. (B) Comparison of the sequences of the PAPOLG genes of Homo sapiens (Hs), Mus musculus (Mm), Gallus gallus (Gg), and Danio rerio (Dr) immediately upstream of the primary poly(A) cleavage site. Shaded sequences denote identity to the human sequence. TGTAN elements are highlighted in bold and outlined with solid boxes; AATAAA-related elements are highlighted in bold and outlined with dashed boxes. Asterisk denotes the poly(A) cleavage site.

Figure 2.

Conserved UGUAN elements within the PAPOLA pre-mRNA bind CFI_m and enhance 3′ processing. (A) Sequence of the 5′ end of the PAPOLA RNAs used for in vitro analysis. Each of the RNAs is identical to the wild-type RNA (PAPαwt) except at the indicated positions. (B) Poly(A) site cleavage. Uniformly ³²P-lableled RNA substrates were incubated in HeLa cell nuclear extract with 3′dATP for 30 min at 30°C, and the RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. (C) Poly(A) addition. Uniformly ³²P-lableled precleaved RNA substrates were incubated in HeLa cell nuclear extract with ATP for 15 min at 30°C, and the RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. (D) The results of four independent poly(A) site cleavage experiments (shaded bars) and four independent poly(A) addition experiments (open bars) are shown as an average, with the SD shown as error bars. Within each experiment, the cleavage and poly(A) addition efficiencies of each of the RNAs is plotted relative to the efficiency of the PAPαwt RNA, which is arbitrarily set to 100%. (E) Gel mobility shift analysis of 3′ processing complexes assembled in HeLa cell nuclear extract. Uniformly ³²P-labeled full-length (lanes 1-5) or precleaved (lanes 6-10) RNAs were incubated with HeLa cell nuclear extract for 5 min at 30°C, followed by the addition of heparin to 5 mg/mL, and the RNA/protein complexes were resolved on a nondenaturing 3% polyacrylamide gel. (F) Gel mobility shift analysis of CFI_m/RNA complexes. Two picomoles of recombinant CFI_m was incubated with uniformly ³²P-labeled precleaved RNAs for 5 min at 30°C and the RNA/protein complexes were resolved on a nondenaturing 3% polyacrylamide gel.

Figure 3.

Conserved UGUAN elements within the PAPOLG pre-mRNA are required for efficient 3′ processing in vitro. (A) Sequence of the 5′ end of the RNAs used for in vitro analysis. Each of the RNAs is identical to the wild-type RNA (PAPγwt) except at the indicated positions. (B) Poly(A) site cleavage. Uniformly ³²P-lableled RNA substrates were incubated in HeLa cell nuclear extract with 3′dATP for 30 min at 30°C, and the RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. (C) Poly(A) addition. Uniformly ³²P-lableled precleaved RNA substrates were incubated in HeLa cell nuclear extract with ATP for 30 min at 30°C, and the RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. Lanes 1-5 were subjected to autoradiography for 15 h, lanes 6-9 for 5 h. (D,E) The results of four independent poly(A) site cleavage experiments (shaded bars) and four independent poly(A) addition experiments (open bars) are shown as an average, with the SD shown as error bars. Within each experiment, the cleavage and poly(A) addition efficiencies of each of the RNAs is plotted relative to the efficiency of the PAPγwt RNA, which is arbitrarily set to 100%.

Figure 4.

UGUAN elements within the PAPOLG pre-mRNA bind CFI_m and contribute to 3′ processing complex assembly. (A) Gel mobility shift analysis of 3′ processing complexes assembled in HeLa cell nuclear extract. Uniformly ³²P-labeled full-length (lanes 1-9) or precleaved (lanes 10-18) RNAs were incubated with HeLa cell nuclear extract for 5 min at 30°C, followed by the addition of heparin to 5 mg/mL, and the RNA/protein complexes were resolved on a nondenaturing 3% polyacrylamide gel. (B) Gel mobility shift analysis of CFI_m/RNA complexes. Two picomoles of recombinant CFI_m was incubated with each uniformly ³²P-labeled precleaved RNA for 5 min at 30°C and the RNA/protein complexes were resolved on a nondenaturing 3% polyacrylamide gel.

Figure 5.

CFI_m contributes to the recruitment of CPSF. (A) The impact of the sequestration of CPSF on poly(A) addition at the Ad-L3, PAPOLG, and PAPOLA poly(A) sites. Uniformly ³²P-labeled precleaved RNA substrates were incubated in HeLa cell nuclear extract in the presence of ATP for 30 min at 30°C. An RNA oligo containing a 23-nt segment of the SV40 late poly(A) site (5′-CUGCAAUAAACAAGUUAACAACA-3′) was added at the indicated concentrations prior to incubation. The reactions in lanes 5, 10, 15, and 20 were incubated with 100 pmol of an RNA oligo of the same sequence, except for two hexamer mutations (AACACA). The RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. (B) The results of four independent sets of poly(A) addition experiments are shown, with the SD at each RNA competitor concentration shown as error bars. Within each experiment, the efficiency of poly(A) addition in the absence of the RNA competitor is arbitrarily set to 100%. (Filled circles) PAPγwt; (filled triangles) PAPαwt; (filled squares) PAPαΔ1/2; (open circles) Ad-L3.

Figure 6.

CFI_m directs sequence-specific, A(A/U)UAAA-independent poly(A) addition through its interaction with hFip1 and poly(A) polymerase. (A) Impact of the addition of recombinant CFI_m on poly(A) addition to the PAPαwt (lanes 1,2), PAPαΔ1/2 (lanes 3,4), PAPγwt (lanes 5,6), and PAPγΔ1/2 (lanes 7,8) RNA substrates in reactions reconstituted with purified HeLa cell CPSF and recombinant poly(A) polymerase. Twenty femtomoles of uniformly ³²P-labeled precleaved RNA substrates was incubated with purified HeLa cell CPSF and recombinant poly(A) polymerase in the presence or absence of 1 pmol of CFI_m and ATP for 30 min at 30°C, and the RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. (B) Coimmunoprecipitation of CFI_m and CPSF. Recombinant hexahis-tagged CFI_m was mixed with HeLa cell nuclear extract and RNase A and subjected to immunoprecipitation with a mouse anti-hexahis antibody (lane 3), an affinity-purified CPSF160K rabbit anti-peptide antibody (lane 6), or control antibodies (mouse IgG1 [lane 2], and rabbit IgG [lane 5]). Aliquots of the immunoprecipitates were analyzed by Western analysis using the anti-CPSF160K antibody (lanes 1-3) or the anti-hexahis antibody (lanes 4-6). (Lane 1) Five percent of the input HeLa cell nuclear extract. (Lane 6) Five percent of the input recombinant CFI_m. (C) Purified baculovirus-expressed recombinant CFI_m and hFip1. Proteins were resolved on an SDS-polyacrylamide gel and stained with Coomassie Blue. (M) Protein standards. (D) Poly(A) addition. Twenty femtomoles of uniformly ³²P-lableled precleaved PAPγwt RNA substrate was incubated with the indicated proteins in the presence of ATP for 60 min at 30°C. Each reaction contained 10 fmol of E. coli-expressed poly(A) polymerase. Lanes 2 and 4 contained 0.5 pmol of hFip1, and lanes 3 and 4 contained 1 pmol of CFI_m. The RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. (E) Twenty femtomoles of the indicated uniformly ³²P-lableled precleaved RNA substrates was incubated as in B with 10 fmol of poly(A) polymerase, 0.5 pmol of hFip1, and 1 pmol of CFI_m. The RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel.

Figure 7.

ChIP analysis of the G6PD gene in HeLa cells. Four regions of the human G6PD gene were subjected to ChIP analysis: (a) a 273-bp sequence located within exon 1, 159 bp downstream of the transcription start site and 15,433 bp upstream of the poly(A) site; (b) a 232-bp sequence located within exon 10, 14,455 bp downstream of the transcription start site and 1177 bp upstream of the poly(A) site; (c) a 227-bp segment located within exon 13, 15,628 bp downstream of the transcription start site and 10 bp upstream of the poly(A) site; and (d) a 265-bp segment located 796 bp downstream of the poly(A) site. (A) A schematic representation of the relative positions of segments of the G6PD gene analyzed by ChIP. (Note that the diagram is not drawn to scale.) (B) ChIP analysis of the human G6PD gene. (mock) Negative control in which buffer was substituted for chromatin. The H14 monoclonal antibody recognizes the RNAPII CTD phosphorylated at Ser 5, the H5 monoclonal antibody recognizes the RNAPII CTD phosphorylated at Ser 2, and the 8WG16 monoclonal antibody recognizes an unphosphorylated RNAPII CTD epitope. The antibodies are described in detail in Materials and Methods. Following agarose gel electrophoresis, PCR products were visualized by ethidium bromide staining.