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C19MC microRNAs are processed from introns of large Pol-II, non-protein-coding transcripts.

Bortolin-Cavaillé ML, Dance M, Weber M, Cavaillé J - Nucleic Acids Res. (2009)

Bottom Line: This 100-kb long cluster consists of 46 tandemly repeated, primate-specific pre-miRNA genes that are flanked by Alu elements (Alus) and embedded within a approximately 400- to 700-nt long repeated unit.It has been proposed that C19MC miRNA genes are transcribed by RNA polymerase III (Pol-III) initiating from A and B boxes embedded in upstream Alu repeats.Here, we show that C19MC miRNAs are intron-encoded and processed by the DGCR8-Drosha (Microprocessor) complex from a previously unidentified, non-protein-coding Pol-II (and not Pol-III) transcript which is mainly, if not exclusively, expressed in the placenta.

View Article: PubMed Central - PubMed

Affiliation: Université de Toulouse, UPS, Laboratoire de Biologie Moléculaire Eucaryote and CNRS, LBME, F-31000 Toulouse, France.

ABSTRACT
MicroRNAs are tiny RNA molecules that play important regulatory roles in a broad range of developmental, physiological or pathological processes. Despite recent progress in our understanding of miRNA processing and biological functions, little is known about the regulatory mechanisms that control their expression at the transcriptional level. C19MC is the largest human microRNA gene cluster discovered to date. This 100-kb long cluster consists of 46 tandemly repeated, primate-specific pre-miRNA genes that are flanked by Alu elements (Alus) and embedded within a approximately 400- to 700-nt long repeated unit. It has been proposed that C19MC miRNA genes are transcribed by RNA polymerase III (Pol-III) initiating from A and B boxes embedded in upstream Alu repeats. Here, we show that C19MC miRNAs are intron-encoded and processed by the DGCR8-Drosha (Microprocessor) complex from a previously unidentified, non-protein-coding Pol-II (and not Pol-III) transcript which is mainly, if not exclusively, expressed in the placenta.

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Related in: MedlinePlus

C19MC-HG as a Pol-II, pri-miRNA transcript. (A) Pol-II is recruited at the endogenously-expressed C19MC locus. Left: miR-515-1, miR-517a and miR-519a-1 genes are endogenously-expressed in JEG3, but not in HeLa or HEK293 cells. miRNA expression was monitored by northern blot with specific oligonucleotide probes. Note that since microRNA genes mapping to C19MC are highly related to each other, causing potential cross-hybridization, it is extremely difficult to formally demonstrate their specific expression by northern blot although stringent hybridization conditions were used. Right: ChIPs were performed in JEG3 and HeLa cells with anti-Pol-II antibodies (H14, Covance) that recognize the phosphorylated CTD serine 5. Antibodies against fibrillarin (Fib) were used as a negative control. C19MC and H2B genes (used as positive control for Pol-II) were simultaneously detected by multiplexed PCR. A representative ethidium bromide-stained agarose gel of three independent ChIPs is shown and miRNA gene loci enriched for RNA Pol-II are shown in Figure 1A. (B) C19MC gene expression is sensitive to α-amanitin. JEG-3 cells were either treated by α-aminitin (left) or actinomycin D (right) as indicated and C19MC microRNA gene expression was assayed by northern blot using a mixture of 32P-labelled oligonucleotides antisense to mature miR-517a, miR-515-1 and miR-519a-1. By probing the membrane with an antisense probe to the mature miR-517 family, the same α-amanitin sensitivity was also specifically demonstrated for expression of mir-517a, one of the two C19MC microRNA genes not preceded by any (TTTT) sequence (not shown). The same membrane was also probed with a tRNATyr and pre-tRNATyr-specific probe that recognizes introns, as well as with a snaR-A specific probe. (C) C19MC-HG transcripts are processed by Microprocessor. Unspliced C19MC-HG expression was monitored by RNAse A/T1 mapping with a mixture of three related 32P-labelled antisense riboprobes to ∼180- to 200-nt long segments of C19MC spanning the pre-miRNA and the surrounding intronic sequences of the three randomly chosen miR-518b, miR-518e and miR-523 gene loci. Left: JEG-3 cells were either treated (+) or not (−) by α-aminitin (20 μg/ml, 9 h). Right: JEG-3 cells were transiently transfected with siRNA-negative control or siRNAs directed against Drosha and DGCR8 mRNAs. HeLa cells were used as a negative control. Probe: ∼2000 c.p.m. of undigested riboprobes. H20: RNAse A/T1 mapping carried out without any RNA. At the bottom of the gel, a shorter exposure is shown for pre-miRNA signals.
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Figure 3: C19MC-HG as a Pol-II, pri-miRNA transcript. (A) Pol-II is recruited at the endogenously-expressed C19MC locus. Left: miR-515-1, miR-517a and miR-519a-1 genes are endogenously-expressed in JEG3, but not in HeLa or HEK293 cells. miRNA expression was monitored by northern blot with specific oligonucleotide probes. Note that since microRNA genes mapping to C19MC are highly related to each other, causing potential cross-hybridization, it is extremely difficult to formally demonstrate their specific expression by northern blot although stringent hybridization conditions were used. Right: ChIPs were performed in JEG3 and HeLa cells with anti-Pol-II antibodies (H14, Covance) that recognize the phosphorylated CTD serine 5. Antibodies against fibrillarin (Fib) were used as a negative control. C19MC and H2B genes (used as positive control for Pol-II) were simultaneously detected by multiplexed PCR. A representative ethidium bromide-stained agarose gel of three independent ChIPs is shown and miRNA gene loci enriched for RNA Pol-II are shown in Figure 1A. (B) C19MC gene expression is sensitive to α-amanitin. JEG-3 cells were either treated by α-aminitin (left) or actinomycin D (right) as indicated and C19MC microRNA gene expression was assayed by northern blot using a mixture of 32P-labelled oligonucleotides antisense to mature miR-517a, miR-515-1 and miR-519a-1. By probing the membrane with an antisense probe to the mature miR-517 family, the same α-amanitin sensitivity was also specifically demonstrated for expression of mir-517a, one of the two C19MC microRNA genes not preceded by any (TTTT) sequence (not shown). The same membrane was also probed with a tRNATyr and pre-tRNATyr-specific probe that recognizes introns, as well as with a snaR-A specific probe. (C) C19MC-HG transcripts are processed by Microprocessor. Unspliced C19MC-HG expression was monitored by RNAse A/T1 mapping with a mixture of three related 32P-labelled antisense riboprobes to ∼180- to 200-nt long segments of C19MC spanning the pre-miRNA and the surrounding intronic sequences of the three randomly chosen miR-518b, miR-518e and miR-523 gene loci. Left: JEG-3 cells were either treated (+) or not (−) by α-aminitin (20 μg/ml, 9 h). Right: JEG-3 cells were transiently transfected with siRNA-negative control or siRNAs directed against Drosha and DGCR8 mRNAs. HeLa cells were used as a negative control. Probe: ∼2000 c.p.m. of undigested riboprobes. H20: RNAse A/T1 mapping carried out without any RNA. At the bottom of the gel, a shorter exposure is shown for pre-miRNA signals.

Mentions: Revisiting microRNA gene organization at C19MC, the largest human microRNA gene cluster. (A) Schematic representation of the ∼100-kb long C19MC (HG18: 58 860 000–58 962 300) mapping at human chromosome 19q13.41. Pre-miRNA genes are symbolized as stem–loop structures. Repeated exons of C19MC-HG are indicated as grey boxes and Alus (as annotated at http://genome.ucsc.edu) are indicated by vertical bars, with green and red bars corresponding to the sense and antisense orientations relative to the pre-miRNA genes, respectively. Horizontal blue bars show miRNA gene loci that have been identified by RNA polymerase-II ChIP experiments described in Figure 3A (the number of sequenced clones is indicated in brackets). (B) Sequence alignment of human exons 1–37. The multiple sequence alignment was generated with Multalin (http://bioinfo.genopole-toulouse.prd.fr/multalin/cgi-bin/multalin.pl) and conserved nucleotides were colored with GeneDoc (http://www.Cris.com/~ketchup/genedoc.shtml). Donor (5′SS) and acceptor (3′SS) splice sites are indicated. Asterisks indicate an alternative 5′SS found in a subset of spliced exons found in cDNA clones we sequenced (not shown). The position of primers 3′-exon(1) and 5′-exon(1) used in Figure 2C are indicated on the top. An LTR sequence inserted in exon 29 has been deleted.


C19MC microRNAs are processed from introns of large Pol-II, non-protein-coding transcripts.

Bortolin-Cavaillé ML, Dance M, Weber M, Cavaillé J - Nucleic Acids Res. (2009)

C19MC-HG as a Pol-II, pri-miRNA transcript. (A) Pol-II is recruited at the endogenously-expressed C19MC locus. Left: miR-515-1, miR-517a and miR-519a-1 genes are endogenously-expressed in JEG3, but not in HeLa or HEK293 cells. miRNA expression was monitored by northern blot with specific oligonucleotide probes. Note that since microRNA genes mapping to C19MC are highly related to each other, causing potential cross-hybridization, it is extremely difficult to formally demonstrate their specific expression by northern blot although stringent hybridization conditions were used. Right: ChIPs were performed in JEG3 and HeLa cells with anti-Pol-II antibodies (H14, Covance) that recognize the phosphorylated CTD serine 5. Antibodies against fibrillarin (Fib) were used as a negative control. C19MC and H2B genes (used as positive control for Pol-II) were simultaneously detected by multiplexed PCR. A representative ethidium bromide-stained agarose gel of three independent ChIPs is shown and miRNA gene loci enriched for RNA Pol-II are shown in Figure 1A. (B) C19MC gene expression is sensitive to α-amanitin. JEG-3 cells were either treated by α-aminitin (left) or actinomycin D (right) as indicated and C19MC microRNA gene expression was assayed by northern blot using a mixture of 32P-labelled oligonucleotides antisense to mature miR-517a, miR-515-1 and miR-519a-1. By probing the membrane with an antisense probe to the mature miR-517 family, the same α-amanitin sensitivity was also specifically demonstrated for expression of mir-517a, one of the two C19MC microRNA genes not preceded by any (TTTT) sequence (not shown). The same membrane was also probed with a tRNATyr and pre-tRNATyr-specific probe that recognizes introns, as well as with a snaR-A specific probe. (C) C19MC-HG transcripts are processed by Microprocessor. Unspliced C19MC-HG expression was monitored by RNAse A/T1 mapping with a mixture of three related 32P-labelled antisense riboprobes to ∼180- to 200-nt long segments of C19MC spanning the pre-miRNA and the surrounding intronic sequences of the three randomly chosen miR-518b, miR-518e and miR-523 gene loci. Left: JEG-3 cells were either treated (+) or not (−) by α-aminitin (20 μg/ml, 9 h). Right: JEG-3 cells were transiently transfected with siRNA-negative control or siRNAs directed against Drosha and DGCR8 mRNAs. HeLa cells were used as a negative control. Probe: ∼2000 c.p.m. of undigested riboprobes. H20: RNAse A/T1 mapping carried out without any RNA. At the bottom of the gel, a shorter exposure is shown for pre-miRNA signals.
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Related In: Results  -  Collection

License
Show All Figures
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Figure 3: C19MC-HG as a Pol-II, pri-miRNA transcript. (A) Pol-II is recruited at the endogenously-expressed C19MC locus. Left: miR-515-1, miR-517a and miR-519a-1 genes are endogenously-expressed in JEG3, but not in HeLa or HEK293 cells. miRNA expression was monitored by northern blot with specific oligonucleotide probes. Note that since microRNA genes mapping to C19MC are highly related to each other, causing potential cross-hybridization, it is extremely difficult to formally demonstrate their specific expression by northern blot although stringent hybridization conditions were used. Right: ChIPs were performed in JEG3 and HeLa cells with anti-Pol-II antibodies (H14, Covance) that recognize the phosphorylated CTD serine 5. Antibodies against fibrillarin (Fib) were used as a negative control. C19MC and H2B genes (used as positive control for Pol-II) were simultaneously detected by multiplexed PCR. A representative ethidium bromide-stained agarose gel of three independent ChIPs is shown and miRNA gene loci enriched for RNA Pol-II are shown in Figure 1A. (B) C19MC gene expression is sensitive to α-amanitin. JEG-3 cells were either treated by α-aminitin (left) or actinomycin D (right) as indicated and C19MC microRNA gene expression was assayed by northern blot using a mixture of 32P-labelled oligonucleotides antisense to mature miR-517a, miR-515-1 and miR-519a-1. By probing the membrane with an antisense probe to the mature miR-517 family, the same α-amanitin sensitivity was also specifically demonstrated for expression of mir-517a, one of the two C19MC microRNA genes not preceded by any (TTTT) sequence (not shown). The same membrane was also probed with a tRNATyr and pre-tRNATyr-specific probe that recognizes introns, as well as with a snaR-A specific probe. (C) C19MC-HG transcripts are processed by Microprocessor. Unspliced C19MC-HG expression was monitored by RNAse A/T1 mapping with a mixture of three related 32P-labelled antisense riboprobes to ∼180- to 200-nt long segments of C19MC spanning the pre-miRNA and the surrounding intronic sequences of the three randomly chosen miR-518b, miR-518e and miR-523 gene loci. Left: JEG-3 cells were either treated (+) or not (−) by α-aminitin (20 μg/ml, 9 h). Right: JEG-3 cells were transiently transfected with siRNA-negative control or siRNAs directed against Drosha and DGCR8 mRNAs. HeLa cells were used as a negative control. Probe: ∼2000 c.p.m. of undigested riboprobes. H20: RNAse A/T1 mapping carried out without any RNA. At the bottom of the gel, a shorter exposure is shown for pre-miRNA signals.
Mentions: Revisiting microRNA gene organization at C19MC, the largest human microRNA gene cluster. (A) Schematic representation of the ∼100-kb long C19MC (HG18: 58 860 000–58 962 300) mapping at human chromosome 19q13.41. Pre-miRNA genes are symbolized as stem–loop structures. Repeated exons of C19MC-HG are indicated as grey boxes and Alus (as annotated at http://genome.ucsc.edu) are indicated by vertical bars, with green and red bars corresponding to the sense and antisense orientations relative to the pre-miRNA genes, respectively. Horizontal blue bars show miRNA gene loci that have been identified by RNA polymerase-II ChIP experiments described in Figure 3A (the number of sequenced clones is indicated in brackets). (B) Sequence alignment of human exons 1–37. The multiple sequence alignment was generated with Multalin (http://bioinfo.genopole-toulouse.prd.fr/multalin/cgi-bin/multalin.pl) and conserved nucleotides were colored with GeneDoc (http://www.Cris.com/~ketchup/genedoc.shtml). Donor (5′SS) and acceptor (3′SS) splice sites are indicated. Asterisks indicate an alternative 5′SS found in a subset of spliced exons found in cDNA clones we sequenced (not shown). The position of primers 3′-exon(1) and 5′-exon(1) used in Figure 2C are indicated on the top. An LTR sequence inserted in exon 29 has been deleted.

Bottom Line: This 100-kb long cluster consists of 46 tandemly repeated, primate-specific pre-miRNA genes that are flanked by Alu elements (Alus) and embedded within a approximately 400- to 700-nt long repeated unit.It has been proposed that C19MC miRNA genes are transcribed by RNA polymerase III (Pol-III) initiating from A and B boxes embedded in upstream Alu repeats.Here, we show that C19MC miRNAs are intron-encoded and processed by the DGCR8-Drosha (Microprocessor) complex from a previously unidentified, non-protein-coding Pol-II (and not Pol-III) transcript which is mainly, if not exclusively, expressed in the placenta.

View Article: PubMed Central - PubMed

Affiliation: Université de Toulouse, UPS, Laboratoire de Biologie Moléculaire Eucaryote and CNRS, LBME, F-31000 Toulouse, France.

ABSTRACT
MicroRNAs are tiny RNA molecules that play important regulatory roles in a broad range of developmental, physiological or pathological processes. Despite recent progress in our understanding of miRNA processing and biological functions, little is known about the regulatory mechanisms that control their expression at the transcriptional level. C19MC is the largest human microRNA gene cluster discovered to date. This 100-kb long cluster consists of 46 tandemly repeated, primate-specific pre-miRNA genes that are flanked by Alu elements (Alus) and embedded within a approximately 400- to 700-nt long repeated unit. It has been proposed that C19MC miRNA genes are transcribed by RNA polymerase III (Pol-III) initiating from A and B boxes embedded in upstream Alu repeats. Here, we show that C19MC miRNAs are intron-encoded and processed by the DGCR8-Drosha (Microprocessor) complex from a previously unidentified, non-protein-coding Pol-II (and not Pol-III) transcript which is mainly, if not exclusively, expressed in the placenta.

Show MeSH
Related in: MedlinePlus