Limits...
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.

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

License
getmorefigures.php?uid=PMC2876958&req=5

f3: 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.

Mentions: To clarify why the MC4R P1 poly(A) site can tolerate mutations in the AUUAAA hexamer, a series of additional plasmids were constructed. Several mutations were introduced into the previously mentioned 23 nucleotides long upstream sequence (Figure 3A: Wt underlined sequence). Interestingly, this 23 nucleotide long sequence is very A-rich and thus resembles somewhat the PE and NUE elements found in yeast and plant 3′end processing sites. Extensive substitutions of adenosines in this sequence with cytidines had no effect on P1 usage as can be seen in the oligo-dT primed RT–PCR analysis presented in Figure 3B (compare lanes 1–3). However, a clear shift from P1 to P2 usage is observed as soon as substantial mutations in the A-rich motif are combined with a point mutation in the AUUAAA hexamer (Figure 3B: lanes 4 and 5). Cleavage at P1 could not be rescued in a construct containing hexamer mutations and where the adenosines in the A-rich motif were substituted by guanosines rather than cytidines (Figure 3C). This suggests that the MC4R poly(A) site contains two potential CPSF-binding sites: an upstream A-rich motif and the AUUAAA. Cleavage and polyadenylation appear to be equally well directed by both of these sequences, which implies that an A-rich motif can functionally substitute for the hexamer and direct cleavage and polyadenylation.


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)

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

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

f3: 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.
Mentions: To clarify why the MC4R P1 poly(A) site can tolerate mutations in the AUUAAA hexamer, a series of additional plasmids were constructed. Several mutations were introduced into the previously mentioned 23 nucleotides long upstream sequence (Figure 3A: Wt underlined sequence). Interestingly, this 23 nucleotide long sequence is very A-rich and thus resembles somewhat the PE and NUE elements found in yeast and plant 3′end processing sites. Extensive substitutions of adenosines in this sequence with cytidines had no effect on P1 usage as can be seen in the oligo-dT primed RT–PCR analysis presented in Figure 3B (compare lanes 1–3). However, a clear shift from P1 to P2 usage is observed as soon as substantial mutations in the A-rich motif are combined with a point mutation in the AUUAAA hexamer (Figure 3B: lanes 4 and 5). Cleavage at P1 could not be rescued in a construct containing hexamer mutations and where the adenosines in the A-rich motif were substituted by guanosines rather than cytidines (Figure 3C). This suggests that the MC4R poly(A) site contains two potential CPSF-binding sites: an upstream A-rich motif and the AUUAAA. Cleavage and polyadenylation appear to be equally well directed by both of these sequences, which implies that an A-rich motif can functionally substitute for the hexamer and direct cleavage and polyadenylation.

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