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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 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.
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f1: 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.

Mentions: As terminal intron removal has long been known to contribute significantly to the efficiency of cleavage and polyadenylation, we addressed how efficient 3′end processing is achieved in naturally intronless genes. For this, we analysed 3′end formation in the human MC4R gene. The analysis of viral and some human intronless genes suggested that these pre-mRNAs generally rely on auxiliary sequences located in the 3′UTR or 3′flanking regions to direct efficient 3′end processing (Huang and Carmichael, 1997; Conrad and Steitz, 2005; Guang and Mertz, 2005; Dalziel et al, 2007). To identify auxiliary cis-elements required for cleavage and polyadenylation of the MC4R pre-mRNA, we used several reporter plasmids. We first cloned 1.3 kb of sequences located downstream of the MC4R stop codon including the 3′UTR, and 1044 nucleotides of 3′flanking sequence into a cytomegalovirus (CMV) promoter-driven reporter gene that contained 5′untranslated region (5′UTR) sequences from the MC1R gene and the green fluorescence protein (GFP) open reading frame (ORF) (Figure 1A).


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

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

f1: 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.
Mentions: As terminal intron removal has long been known to contribute significantly to the efficiency of cleavage and polyadenylation, we addressed how efficient 3′end processing is achieved in naturally intronless genes. For this, we analysed 3′end formation in the human MC4R gene. The analysis of viral and some human intronless genes suggested that these pre-mRNAs generally rely on auxiliary sequences located in the 3′UTR or 3′flanking regions to direct efficient 3′end processing (Huang and Carmichael, 1997; Conrad and Steitz, 2005; Guang and Mertz, 2005; Dalziel et al, 2007). To identify auxiliary cis-elements required for cleavage and polyadenylation of the MC4R pre-mRNA, we used several reporter plasmids. We first cloned 1.3 kb of sequences located downstream of the MC4R stop codon including the 3′UTR, and 1044 nucleotides of 3′flanking sequence into a cytomegalovirus (CMV) promoter-driven reporter gene that contained 5′untranslated region (5′UTR) sequences from the MC1R gene and the green fluorescence protein (GFP) open reading frame (ORF) (Figure 1A).

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