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

Nunes NM, Li W, Tian B, Furger A - EMBO J. (2010)

Bottom Line: Mutation of the AUUAAA hexamer had little effect on MC4R 3'end processing but small changes in the short DSE severely reduced cleavage efficiency.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford, UK.

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.

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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.
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f4: 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.

Mentions: To further analyse the role of individual adenosines in the A-rich upstream region, we constructed additional plasmids that contained single A to G changes in this area in the double hexamer mutant (h1h2*) background (Figure 4A: h1h2*-a1–8). For this RP analysis we used a ‘composite' antisense riboprobe that can be used for all mutant constructs because it does not contain the regions in which sequence changes were introduced. The sequence of this antisense riboprobe corresponds to a direct fusion of the GFP ORF to the MC4R core poly(A) signal and 200 nt of 3′flanking sequences (Figure 4A). The probe results in two protected bands, a 240 nucleotide band representing total reporter transcripts and a second 100 nucleotide long protected band that will only be present if transcripts escape cleavage at P1 and are subsequently either processed at P2 or readthrough P2 (Figure 4A).


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

Nunes NM, Li W, Tian B, Furger A - EMBO J. (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2876958&req=5

f4: 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.
Mentions: To further analyse the role of individual adenosines in the A-rich upstream region, we constructed additional plasmids that contained single A to G changes in this area in the double hexamer mutant (h1h2*) background (Figure 4A: h1h2*-a1–8). For this RP analysis we used a ‘composite' antisense riboprobe that can be used for all mutant constructs because it does not contain the regions in which sequence changes were introduced. The sequence of this antisense riboprobe corresponds to a direct fusion of the GFP ORF to the MC4R core poly(A) signal and 200 nt of 3′flanking sequences (Figure 4A). The probe results in two protected bands, a 240 nucleotide band representing total reporter transcripts and a second 100 nucleotide long protected band that will only be present if transcripts escape cleavage at P1 and are subsequently either processed at P2 or readthrough P2 (Figure 4A).

Bottom Line: Mutation of the AUUAAA hexamer had little effect on MC4R 3'end processing but small changes in the short DSE severely reduced cleavage efficiency.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford, UK.

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.

Show MeSH