A functional human Poly(A) site requires only a potent DSE and an A-rich upstream sequence

Abstract

We have analysed the sequences required for cleavage and polyadenylation in the intronless melanocortin 4 receptor (MC4R) pre-mRNA. Unlike other intronless genes, 3'end processing of the MC4R primary transcript is independent of any auxiliary sequence elements and only requires the core poly(A) sequences. Mutation of the AUUAAA hexamer had little effect on MC4R 3'end processing but small changes in the short DSE severely reduced cleavage efficiency. The MC4R poly(A) site requires only the DSE and an A-rich upstream sequence to direct efficient cleavage and polyadenylation. Our observation may be highly relevant for the understanding of how human noncanonical poly(A) sites are recognised. This is supported by a genome-wide analysis of over 10 000 poly(A) sites where we show that many human noncanonical poly(A) signals contain A-rich upstream sequences and tend to have a higher frequency of U and GU nucleotides in their DSE compared with canonical poly(A) signals. The importance of A-rich elements for noncanonical poly(A) site recognition was confirmed by mutational analysis of the human JUNB gene, which contains an A-rich noncanonical poly(A) signal.

Figure 1

The MC4R poly(A) site does not require auxiliary sequence elements. (A) Diagram depicting the MC4R reporter genes: F and deletion of 3′flanking sequences (FΔ1, FΔ2 and FΔ3). Vertical arrows indicate end of the deletion clones relative to Wt sequence. The borders between ORF, 3′UTR, 3′flank and vector backbone are indicated by thin straight vertical lines. Promoter (CMV) and the GFP ORF are represented by open boxes, lines across indicate that regions are not drawn to scale. MC4R poly(A) sites P1 and P2 are filled triangles. The regions deleted in clones ΔU1, ΔU2 and Δ23 are indicated below the graph. RP fragments uncleaved (rt), cleaved at P1 (P1) or cleaved at P2 (P2) are shown as dotted lines and the expected lengths are indicated. The positions of the F1 and F2 forward primers used in the RT–PCR analysis shown in (C) and (D), respectively, are shown above the diagram. (B) RP analysis of total RNA isolated from HEK293 cells transiently transfected with constructs containing 3′flank deletions. Transcripts not cleaved at P1 are indicated either as transcripts cleaved at P2 for (F, FΔ1) or uncleaved readthrough transcripts rt=rt(F, FΔ1), rt(FΔ2), rt(FΔ3). Alternative cleavage site used at P1 observed with plasmids FΔ2 and FΔ3 is indicated by (^*). FΔ1 is subsequently referred to as wild-type (Wt) (C, D) RT–PCR analysis of constructs containing deletions in the 3′UTR. RT–PCR products corresponding to mRNAs cleaved at either P1 or P2 are indicated for Wt and UTR deletion clones. Size markers are indicated.

Figure 2

Mutations of the core poly(A) sequences have unexpected effects on cleavage efficiency. (A) Diagram depicting the MC4R reporter gene where both potential poly(A) sites are indicated by filled triangles. Potential priming of oligo-dT reverse primers (dT) at P1 and P2 and forward primer (F1) is depicted above the diagram. The sequences surrounding P2 are indicated in the box above the graph. The hexamer is in bold, nucleotides of the DSE are underlined and the site of cleavage is indicated by the open triangle. Below the diagram is the Wt sequence surrounding P1, the two hexamers are in bold and indicated by (H1) and (H2), respectively. Capital H represents clones with wild-type hexamer sequences, small letters h (h1 and/or h2) represent mutated hexamers. Open triangle marks the site of cleavage at P1 and the DSE (D) is indicated in bold letter. The mutated nucleotides for each clone are depicted in bold and underlined below the Wt sequence. (B–D) RP (top gels) and RT–PCR analysis (bottom gels) of total RNA isolated from transiently transfected cells. Expected migration patterns of transcripts cleaved at P1, P2 or unprocessed readthrough RNA (rt) are indicated on the left of each gel. Quantitations of independent RPs as an average percentage of total P1 readthrough transcripts are shown below.

Figure 3

Mutations in the A-rich sequence and the hexamer are required to inactivate MC4R P1. (A) The diagram of the MC4R reporter gene and the Wt sequence surrounding the P1 poly(A) site is shown, underlined letters represent the 23 nucleotides long upstream sequence. The open triangle marks the site of cleavage at P1 and the DSE (D) is indicated in bold. The changed nucleotides in each construct are shown in bold and underlined letters below the Wt sequence. The four UGUAN motifs located in the 3′UTR are indicated by (4x) UGUAN in the diagram. Forward primers (F1 and F2) and sites of potential reverse priming by oligo-dT at P1 and P2, respectively, are shown above the diagram. (B–D) Qualitative oligo-dT primed RT–PCR analysis of total RNA isolated from HEK 293 cells transiently transfected with the Wt and mutant plasmids. F1 or F2 use is indicated on top right of gels.

Figure 4

The MC4R DSE only requires an A-rich upstream sequence for efficient cleavage. (A) Diagram of the MC4R reporter is shown and the details are as in Figure 3. The outline of the composite RP probe is depicted above and the protected fragments are shown as dotted lines. All transcripts result in a 240 nt protected band Tot (P1+P2+P2rt) and transcripts not cleaved at P1 (P2+P2rt) give an additional protected band rt (100 nt). The sequences surrounding P1 are shown below and the nucleotide substitutions for each clone are indicated in bold and underlined letters below the Wt sequence. (B–D) RPs of constructs with mutated core sequences and 17A-substitutions. The position of the protected bands is indicated by horizontal arrows at the right of the gels: total transcripts=Tot, transcripts not processed at P1=rt. Quantitation of at least three independent RPs for each clone for each gel is given below the gels. Average percentage of the total transcripts that are not cleaved at P1 is set to 5% for the wild type as determined in Figure 2.

Figure 5

Systematic analysis of poly(A) sites with A(A/U)UAAA and A-rich elements. A-rich elements are hexamers containing at least five As excluding AAUAAA and not overlapping with A(A/U)UAAA. (A) Difference in nucleotide frequency surrounding poly(A) sites with A-rich elements vs A(A/U)UAAA poly(A) sites. (B) Significance of bias of 4-mers in the +5 to +40 nt region of A-rich and A(A/U)UAAA poly(A) sites. A significance score was calculated for each 4-mer based on its bias of occurrence in A-rich or A(A/U)UAAA poly(A) sites using Fisher's exact test (see Materials and methods for detail). The significance score is −log(P-value) if the 4-mer is biased to A(A/U)UAAA poly(A) sites, or log(P-value) if biased to A-rich poly(A) sites. The distribution of significance scores is shown in a histogram. The top 10 4-mers significantly biased to A-rich poly(A) sites and to A(A/U)UAAA poly(A) sites are listed, together with their P-values. The most significant 4-mer, UUUU, is indicated in the histogram. (C) Schematics of single poly(A) sites (S); first (F), middle (M) and last (L) poly(A) sites in genes with alternative poly(A) sites located in the 3′-most exon. Poly(A) sites are indicated by arrows. CDS, coding sequence. (D) Percent of poly(A) sites with A(A/U)UAAA and/or A-rich elements in the −40 to −10 nt region for the 4 poly(A) site types shown in (C). (E) Percent of poly(A) sites with co-occurrence of A(A/U)UAAA or A-rich elements and U-rich (left) or GU-rich elements (right) for the four poly(A) site types. The U-rich or GU-rich sequence elements are described in ‘Materials and methods'. The error bars are standard deviations. The differences in occurrence of U-rich or GU-rich sequence elements were evaluated by Fisher's exact test. Significant ones are indicated by one asterisk (P<0.05) or two asterisks (P<0.01). (F) Percent of poly(A) sites conserved in mouse with co-occurrence of A-rich elements only or A(A/U)UAAA only and downstream GU-rich and/or U-rich elements for the four poly(A) site types. The error bars are standard deviations.

Figure 6

The JUNB pre-mRNAs require an A-rich upstream sequence for efficient cleavage and polyadenylation. (A) Diagram showing the JUNB reporter gene. Origins of the sequences in the plasmid are indicated. JUNB 3′UTR and 3′flanking regions are represented by a dotted line and the graph shows how JUNB sequences are inserted into the MC4R background. The position of the JUNB A-rich region (A), DSE (D) is indicated and the poly(A) sites are represented by JpA and P2, respectively. Lengths of protected RP bands are shown above the graph. All transcripts result in a 240 nucleotide protected band (Tot) and transcripts not cleaved at JpA (rt) give an additional protected band 88 nucleotides in length. The JUNB-specific probe used in (B) is shown as thin black line below the dotted line. The sequences surrounding the JUNB (JpA) cleavage site are shown below the graph and the nucleotide substitutions for each clone are indicated in bold and underlined letters below the Wt sequences (J-Wt). (B, C) RP of total RNA isolated from cells transfected with JUNB Wt and mutant plasmids. Quantitation (n=3) is presented as fold increase of transcripts that are not cleaved at JpA.