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

2010; Springer Nature; Volume: 29; Issue: 9 Linguagem: Inglês

10.1038/emboj.2010.42

1460-2075

Nuno M. Nunes, Wencheng Li, Bin Tian, André Furger,

RNA regulation and disease

Article25 March 2010Open Access A functional human Poly(A) site requires only a potent DSE and an A-rich upstream sequence Nuno Miguel Nunes Nuno Miguel Nunes Laboratory of Genes and Development, Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Wencheng Li Wencheng Li Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Bin Tian Bin Tian Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author André Furger Corresponding Author André Furger Laboratory of Genes and Development, Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Nuno Miguel Nunes Nuno Miguel Nunes Laboratory of Genes and Development, Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Wencheng Li Wencheng Li Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Bin Tian Bin Tian Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author André Furger Corresponding Author André Furger Laboratory of Genes and Development, Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Author Information Nuno Miguel Nunes1, Wencheng Li2, Bin Tian2 and André Furger 1 1Laboratory of Genes and Development, Department of Biochemistry, University of Oxford, Oxford, UK 2Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, NJ, USA *Corresponding author. Laboratory of Genes and Development, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Tel.: +44 0 1865 613 261; Fax: +44 0 1865 613 276; E-mail: [email protected] The EMBO Journal (2010)29:1523-1536https://doi.org/10.1038/emboj.2010.42 There is a Have you seen ...? (May 2010) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction 3′end formation is a fundamental processing step for the maturation of mRNAs in eukaryotes. All protein encoding primary transcripts are cleaved at their 3′end and, with the exception of replication-dependent metazoan histone genes, are subsequently subjected to polyadenylation resulting in mature transcripts with characteristic poly(A) tails. (Proudfoot et al, 2002; Soller, 2006). Cleavage and polyadenylation define critical biochemical events in eukaryotic gene expression promoting transcription termination, terminal intron removal, nuclear–cytoplasmic transport, translation initiation and stability of the mRNA (Proudfoot et al, 2002; Proudfoot, 2004). It is therefore not surprising that 3′end formation represents a crucial regulatory step in eukaryotic gene expression and improper 3′end processing of pre-mRNAs is associated with a number of human diseases (Danckwardt et al, 2008). The co-transcriptional cleavage and polyadenylation reaction is directed by a large multi-protein complex, which in humans can constitute more than 80 proteins (Shi et al, 2009). The key subunits of this large complex are evolutionarily highly conserved and in mammals are represented by four multi-protein components, the cleavage and polyadenylation specificity factor (CPSF), the cleavage stimulation factor (CstF), cleavage factors I and II (CFIm, CFIIm) and the single polypeptide poly(A) polymerase (PAP). In mammals, assembly of the 3′end processing complex is initiated by the cooperative interaction of CPSF and CstF with specific core sequences on the pre-mRNA (Zhao et al, 1999; Proudfoot et al, 2002). This core poly(A) sequence is a bipartite sequence element, which in humans, in 70–80%, is defined by a conserved canonical AAUAAA or AUUAAA upstream hexamer motif, recognised by CPSF, and a somewhat less defined downstream U- or GU-rich region (DSE) contacted by CstF (Zarudnaya et al, 2003; Tian et al, 2005). In vitro, these initial contacts are critical for the recruitment of additional key factors including CFIm, CFIIm and PAP. The efficiency of assembly and subsequent processing is strongly influenced by both the core sequence composition and the spatial arrangement (Zhao et al, 1999). In addition, in many mammalian genes, further auxiliary sequence elements that influence complex assembly can be found both upstream (USEs) and downstream of the core sequences. Furthermore, extensive protein–protein interactions between components of the splicing machinery directing terminal intron removal and the cleavage and polyadenylation apparatus result in reciprocal enhancement of both processing reactions (Vagner et al, 2000; Millevoi et al, 2002, 2006; Kyburz et al, 2006). The cleavage and polyadenylation machineries of plants, yeast and mammals share many homologies (Hunt, 2008) but the sequence elements directing the assembly of the processing complexes differ in parts. Although the canonical A(A/U)UAAA hexamer sequences can be found upstream of the cleavage sites in both yeast and plants, there appears to be fewer constraints regarding their sequence composition. The hexamer-like sequences found in the near upstream region (NUE) in plants and the positioning element (PE) in yeast are often degenerated to little more than A-rich sequences (Zhao et al, 1999; Hunt, 2008). This is in stark contrast to mammalian hexamer sequences that are generally highly intolerant to sequence alterations (Wickens and Stephenson, 1984; Sheets et al, 1990). Interestingly, 20–30% of human genes do not contain canonical hexamers and 3′end processing is directed by noncanonical sequences (Zarudnaya et al, 2003; Tian et al, 2005). In addition, noncanonical poly(A) sites appear to be more frequent in genes that undergo alternative cleavage and polyadenylation (Tian et al, 2005 and this study) suggesting that they may have a critical role in this process. As about half of all human protein encoding genes undergo alternative cleavage and polyadenylation, it is somewhat surprising that we currently only have a poor understanding how noncanonical poly(A) sites are recognised and regulated. It has been shown that at least in some cases the interaction of additional core factors, such as CFIm with UGUAN motifs located in the 3′UTR, can promote cleavage and polyadenylation at such 3′end processing sites (Venkataraman et al, 2005). Interestingly, the sequence composition of these noncanonical poly(A) sites was proposed to be much more similar to those described for plants and yeast (Venkataraman et al, 2005). Similar to noncanonical poly(A) sites, 3′end processing of mammalian and viral intronless primary transcripts also appears to require additional auxiliary cis-elements, perhaps compensating for the lack of enhancement by the splicing factors observed in spliced genes. Several so-called pre-mRNA processing enhancer elements (PPE) located upstream of the core poly(A) sequences have been described for many viral and mammalian non-spliced genes (Huang and Carmichael, 1997; Conrad and Steitz, 2005; Guang and Mertz, 2005). In this work we analysed how 3′end processing is regulated in the human melanocortin 4 receptor gene (MC4R). MC4R is a 333 amino acid 7 transmembrane domain protein encoded by a single exon gene. The MC4R gene is expressed in multiple sites in the brain (Liu et al, 2003) and mutations in this gene have been associated with obesity (Huszar et al, 1997). Our results show that 3′end processing in the intronless MC4R primary transcript, unlike other intronless genes, does not require any auxiliary sequence elements located either in the 3′UTR or in the 3′flanking regions. Optimal cleavage at the MC4R poly(A) site relies solely on the core poly(A) sequences. The relatively short MC4R DSE is shown to be the most critical element and is able to direct efficiently 3′end cleavage independent of the upstream AUUAAA hexamer. We show that the MC4R DSE, similar to both yeast and plant poly(A) sites, only requires an A-rich sequence upstream of the cleavage site for optimal 3′end processing. Our bioinformatics analysis highlights the significance of this finding by showing that many noncanonical poly(A) sites contain A-rich upstream sequences. Importantly, this analysis further shows that such A-rich 3′end processing sites correlate with an increased frequency of U/GU sequences in their DSE compared with poly(A) sites that contain the canonical A(A/U)UAAA hexamers. These observations could be significant for the understanding of how noncanonical poly(A) sites are recognised by the 3′end processing machinery. Indeed, we show that the A-rich sequence in the JUNB pre-mRNA is critical for cleavage and polyadenylation at its noncanonical poly(A) site. Thus, we propose that many noncanonical poly(A) sites may, similar to MC4R, predominantly rely on a potent DSE mediating a tight interaction with CstF. This interaction may in turn be critical to facilitate recruitment of CPSF to less favourable A-rich upstream sequences and so afford efficient 3′end processing of pre-mRNAs in the absence of canonical hexamers. Results MC4R pre-mRNA 3′end formation does not require additional sequence elements located in the 3′flank or 3′UTR 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). 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. Download figure Download PowerPoint This original MC4R reporter plasmid was transfected into HEK 293 cells and total RNA was subsequently isolated and analysed by RNAse protection (RP) to map the MC4R 3′end processing site. This was necessary because the poly(A) site of MC4R is not annotated and sequence comparison shows at least two potential 3′end processing sites located in the first 400 nucleotides downstream of the MC4R stop codon (Figure 1A: P1, P2). Our analysis mapped the poly(A) cleavage site to a position 292 nucleotides downstream of the MC4R ORF (Figure 1A: P1). This initial mapping of the processing site was subsequently confirmed by 3′RACE (data not shown). It is worth noting that we also confirmed that a hexamer overlapping the endogenous MC4R stop codon and the first nucleotides within the 3′UTR is not functional (Supplementary Figure 1: A and B). The above described RP analysis also showed that the first poly(A) site (P1) is efficiently used and no readthrough transcripts either processed at the second poly(A) site (P2) or not processed at all (rt) were detected (Figure 1B: lane 2: P2, rt). To verify the presence of potential auxiliary sequence elements in the 3′flank we constructed and analysed three additional plasmids with gradually shorter 3′flanking sequences compared with the full-length clone F (Figure 1A: FΔ1, FΔ2, FΔ3). For the RP analysis, we used an antisense riboprobe complementary to sequences overlapping P1 and P2, which results in protected bands of the same length for all clones representing transcripts cleaved at P1. Note that because of the deletions, transcripts from the FΔ2 and FΔ3 plasmids, which are not processed at P1, would result in protected readthrough bands of different lengths compared with transcripts originating from the F and FΔ1 plasmids (rt(F, FΔ1), rt(FΔ2) and rt(FΔ3)). As can be seen in Figure 1B, deletion of all but 25 nucleotides of the 3′flank (FΔ3, counted from the site of cleavage) or less (FΔ2, FΔ1) had no effect on the cleavage efficiency at P1 because no bands can be seen corresponding to transcripts that failed to cleave at P1 (Figure 1B: compare lanes 2 and 3 and lanes 5–7, rt(F, FΔ1), rt(FΔ3), rt(FΔ2) and P2, respectively). However, large deletions of the 3′flank resulted in the appearance of an additional less intense protected band that is likely to be caused by a shift in the site of cleavage in some of the FΔ3 and FΔ2 transcripts (Figure 1B: lanes 6 and 7; *). For all further experiments, we used FΔ1 as the wild-type reference and thus FΔ1 is subsequently referred to as Wt. We next addressed whether sequences located in the 3′UTR are required for efficient 3′end processing of the MC4R pre-mRNA. To that end, we constructed a plasmid that had almost all 3′UTR sequences removed, retaining only the last 23 nucleotides immediately upstream of the P1 AUUAAA hexamer and a second plasmid that had the AUUAAA directly fused to the GFP stop codon (Figure 1A: ΔU1, ΔU2, respectively). As can be seen in Figure 1C, oligo-dT primed RT–PCR analysis of total RNA isolated from transfected cells showed that ΔU1 only had a marginal effect on P1 usage. In contrast, the deletion of the entire 3′UTR (ΔU2) appeared to dramatically shift the preferred cleavage site from P1 to P2. This initial result suggested that, there is either a 23 nucleotide long enhancer element located immediately upstream of the P1 hexamer or that locating P1 close to the GFP ORF or 5′UTR sequences somehow reduces processing efficiency at the P1 poly(A) site. To clarify this, a third construct was built that retained all but the last 23 nucleotides of the 3′UTR (Figure 1A: Δ23). As can be seen in Figure 1D, deletion of these nucleotides did not result in a significant shift from P1 into P2 usage (compare lanes 1 and 2). From this analysis we concluded that no sequences in the 3′UTR or 3′flank are required to direct efficient cleavage at the MC4R poly(A) site. This analysis also showed that P2 is an additional functional poly(A) site and that transcripts not cleaved at the P1 poly(A) site are subsequently efficiently cleaved at P2. The switch of cleavage at P1 to cleavage at P2 was thus used to measure effects on poly(A) cleavage at P1. Analysis of the MC4R P1 core poly(A) signal The above described analysis suggested that 3′end processing of the MC4R pre-mRNA is directed by nucleotides including and surrounding the core poly(A) sequences. To address the functional relevance of the core sequences, we first focused on the hexamer motifs. The P1 poly(A) site in MC4R contains two potential hexamers, AAGAAA and AUUAAA (see H1 and H2 in Figure 2A). Although the presence of a guanosine at position 3 in hexamers is normally considered as a potent inactivating mutation (Wilusz and Shenk, 1988; Natalizio et al, 2002), it has been shown to be active in at least one gene (Anand et al, 1997). Thus, reporter constructs were created with mutations destroying either (H1h2, h1H2,) or both (h1h2) hexamers (Figure 2A). Cells were transfected with these plasmids and total RNA was subsequently analysed by RP and RT–PCR. Although mutations of hexamer sequences have long been known to severely impair 3′end processing (Conway and Wickens, 1987; Wilusz et al, 1989; Sheets et al, 1990), mutating the MC4R hexamer(s) surprisingly had little effect on cleavage at P1 when analysed by RP and RT–PCR (Figure 2B: compare lanes 1–4 in RP and RT–PCR panels). As the hexamer mutations produced unexpected results, we next created two constructs where two or four uridines in the DSE were substituted by cytidines (Figure 2A: d2, d4 respectively). In contrast to mutations of the hexamers, DSEs are generally described as being relatively tolerant to sequence changes (Zhao et al, 1999). 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. Download figure Download PowerPoint Unlike the hexamer mutations described above, substitution of two uridines resulted in a more than 7-fold increase in P2 usage and introduction of two further substitutions caused a significant 11-fold increase in transcripts cleaved at P2 (Figure 2C). Combining the four U to C substitutions in the DSE with hexamer mutations resulted in a five- to seven-fold increase in P2 usage compared with the hexamer mutations alone, again highlighting the importance of the DSE for the recognition of the MC4R P1 poly(A) site (Figure 2D). From these experiments, we concluded that the MC4R DSE is the critical core sequence element for cleavage and polyadenylation and that this DSE does not require a canonical hexamer upstream of the cleavage site to direct efficient 3′end processing. Mutations in an A-rich upstream sequence and the AUUAAA are necessary to inactivate the MC4R P1 poly(A) site 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. 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. Download figure Download PowerPoint Interestingly, a possible molecular mechanism explaining how A-rich noncanonical poly(A) sites can be recognised by the poly(A) machinery has recently been presented. The PAPOLG pre-mRNA lacks a canonical hexamer but instead contains a critical A-rich sequence upstream of the cleavage site. The recognition and function of this poly(A) site was shown to be dependent on UGUAN motifs located in the 3′UTR and the A-rich upstream sequence. The UGUAN motifs were shown to be recognised by CFIm and this interaction facilitated the recruitment of CPSF and PAP to the pre-mRNA in the absence of a canonical hexamer (Venkataraman et al, 2005). Therefore, similar to PAPOLG, the presence of four UGUAN sequences in the MC4R 3′UTR could explain why the mutation of the AUUAAA hexamer was tolerated in our earlier experiments. To clarify this issue, G to C substitutions were introduced into the four UGUAN elements present in the MC4R 3′UTR in the wild-type plasmid and in the h1h2 construct (Figure 3A: UCUAN-Wt, UCUAN-h1h2). It is important to note that the UCUAN sequence significantly reduces interaction with CFIm in vitro (Venkataraman et al, 2005). Total RNA isolated from transiently transfected cells was analysed by oligo-dT primed RT–PCR. These results show that mutations of the UGUAN elements present in the MC4R 3′UTR had no significant effects on MC4R 3′end processing efficiency at P1 either in the presence or in the absence of a defined hexamer (Figure 3D: compare lanes 1–4: P1, P2). From these observations, we concluded that the UGUAN elements present in the MC4R 3′UTR do not have a functional role in MC4R 3′end formation and that the MC4R poly(A) site only requires a strong DSE and an upstream A-rich region for efficient cleavage and polyadenylation. A potent DSE downstream of a stretch of adenosines is sufficient to direct 3′end cleavage 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). 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. Download figure Download PowerPoint The use of this composite probe confirmed the earlier results, that mutations of upstream hexamers in the MC4R poly(A) site had little effect on P1 usage (Figure 4B: compare lanes 1–3; Figure 4C: lanes 1 and 2). In contrast, mutations affecting the hexamers and/or the DSE reduced P1 usage dramatically, which is shown by the appearance of a second protected band representing transcripts that are not cleaved at P1 (Figure 4B: compare lanes 1–8 with lane 9; Figure 4C: lanes 3 and 4). The overall effects of mutating individual adenosines in the h1h2* background compared with h1h2-d4 clone were modest (Figure 4B: lanes 4–9). Interestingly, mutations of adenosines, which disrupt the stretches of five or four consecutive adenosines surrounding H1 (Figure 4A), consistently affected cleavage at P1 more severely than changing adenosines at the periphery of either stretch (Figure 4B: compare rt in lanes 4 and 8 with 5–7). These results suggested that the adenosines in the A-rich region are unlikely to be part of a novel hexamer-like structure and that uninterrupted stretches of adenosines may be the most critical feature of this region. To test this hypothesis, we created a plasmid in which both the A-rich region and the AUUAAA were replaced with a stretch of 17 adenosines. From this parental 17A construct two more plasmids were created that contained G to C mutations inactivating the four UGUAN motifs present in the 3′UTR and/or with the d4 mutations inactivating the DSE. A control plasmid containing a stretch of cytidines instead of adenosines was also created (Supplementary Figure 1C). The use of the composite probe for the analysis of the 17A plasmids was essential as this probe avoids an experimental artefact that was observed with mutant-specific antisense probes encompassing a long A-stretch. The U-rich sequence in such probes can hybridise with the poly(A) tails of potentially all mRNAs. This can result in protected bands that mimic a cleavage event immediately upstream of the A-rich sequence (data not shown). A similar problem can arise from the use of oligo-dT primed RT–PCR, as miss-priming at the 17 nt A-stretch by oligo-dT (17T) readily occurred and resulted in a slightly faster migrating band (