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A competitive regulatory mechanism discriminates between juxtaposed splice sites and pri-miRNA structures.

Mattioli C, Pianigiani G, Pagani F - Nucleic Acids Res. (2013)

Bottom Line: Moreover, small interfering-mediated silencing of Drosha and/or DGCR8 improves splicing efficiency and abolishes miR-34b production.Thus, the processing of this 3' SO miRNA is regulated in an antagonistic manner by the Microprocessor and the spliceosome owing to competition between these two machineries for the nascent transcript.We propose that this novel mechanism is commonly used to regulate the relative amount of SO miRNA and messenger RNA produced from primary transcripts.

View Article: PubMed Central - PubMed

Affiliation: Human Molecular Genetics, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149, Trieste, Italy.

ABSTRACT
We have explored the functional relationships between spliceosome and Microprocessor complex activities in a novel class of microRNAs (miRNAs), named Splice site Overlapping (SO) miRNAs, whose pri-miRNA hairpins overlap splice sites. We focused on the evolutionarily conserved SO miR-34b, and we identified two indispensable elements for recognition of its 3' splice site: a branch point located in the hairpin and a downstream purine-rich exonic splicing enhancer. In minigene systems, splicing inhibition owing to exonic splicing enhancer deletion or AG 3'ss mutation increases miR-34b levels. Moreover, small interfering-mediated silencing of Drosha and/or DGCR8 improves splicing efficiency and abolishes miR-34b production. Thus, the processing of this 3' SO miRNA is regulated in an antagonistic manner by the Microprocessor and the spliceosome owing to competition between these two machineries for the nascent transcript. We propose that this novel mechanism is commonly used to regulate the relative amount of SO miRNA and messenger RNA produced from primary transcripts.

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Splicing of SO miR-34b requires a purine-rich ESE. Schematic representation of the pcDNA3pY7 miR-34b mutants of the exon. The lines and black box correspond to the intronic miR-34b hairpin and exonic sequences, respectively. The mutated nucleotides of ESEmut are indicated. Lower panel shows the splicing pattern of pcDNA3pY7 miR-34b exonic mutants after transfection in HeLa cells. The identity of the bands is depicted on the right. Numbers below the panel indicate the percentage of splicing expressed as mean±SD of at least three independent experiments.
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gkt614-F4: Splicing of SO miR-34b requires a purine-rich ESE. Schematic representation of the pcDNA3pY7 miR-34b mutants of the exon. The lines and black box correspond to the intronic miR-34b hairpin and exonic sequences, respectively. The mutated nucleotides of ESEmut are indicated. Lower panel shows the splicing pattern of pcDNA3pY7 miR-34b exonic mutants after transfection in HeLa cells. The identity of the bands is depicted on the right. Numbers below the panel indicate the percentage of splicing expressed as mean±SD of at least three independent experiments.

Mentions: To identify additional splicing regulatory elements, we focused on downstream exonic sequences. In several cases, exonic regulatory sequences act on alternative splicing regulating the 3′ ss recognition (42,43). We prepared minigenes with progressive deletions of the exonic sequences. Minigenes 2482, 2469, 2457 and 2436 (Figure 4) contain progressive deletions of 23, 36, 48 and 69 bp of the exon, respectively. Deletion of the last 23 nt of the exon (mutant 2482) reduced the splicing efficiency to 38%, whereas further deletions completely abolished splicing (mutants 2469, 2457 and 2436; Figure 4). To map the regulatory element involved, we made internal 25 bp deletions (Figure 4), and the results showed that the sequence deleted in mutant Δ2457–2482 was sufficient to abolish splicing. This region contains a 9 bp purine-rich GAGAGAAGA sequence, and substitution of the four adenines into pyrimidines induced nearly complete intron retention (ESEmut, Figure 4).Figure 4.


A competitive regulatory mechanism discriminates between juxtaposed splice sites and pri-miRNA structures.

Mattioli C, Pianigiani G, Pagani F - Nucleic Acids Res. (2013)

Splicing of SO miR-34b requires a purine-rich ESE. Schematic representation of the pcDNA3pY7 miR-34b mutants of the exon. The lines and black box correspond to the intronic miR-34b hairpin and exonic sequences, respectively. The mutated nucleotides of ESEmut are indicated. Lower panel shows the splicing pattern of pcDNA3pY7 miR-34b exonic mutants after transfection in HeLa cells. The identity of the bands is depicted on the right. Numbers below the panel indicate the percentage of splicing expressed as mean±SD of at least three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3794580&req=5

gkt614-F4: Splicing of SO miR-34b requires a purine-rich ESE. Schematic representation of the pcDNA3pY7 miR-34b mutants of the exon. The lines and black box correspond to the intronic miR-34b hairpin and exonic sequences, respectively. The mutated nucleotides of ESEmut are indicated. Lower panel shows the splicing pattern of pcDNA3pY7 miR-34b exonic mutants after transfection in HeLa cells. The identity of the bands is depicted on the right. Numbers below the panel indicate the percentage of splicing expressed as mean±SD of at least three independent experiments.
Mentions: To identify additional splicing regulatory elements, we focused on downstream exonic sequences. In several cases, exonic regulatory sequences act on alternative splicing regulating the 3′ ss recognition (42,43). We prepared minigenes with progressive deletions of the exonic sequences. Minigenes 2482, 2469, 2457 and 2436 (Figure 4) contain progressive deletions of 23, 36, 48 and 69 bp of the exon, respectively. Deletion of the last 23 nt of the exon (mutant 2482) reduced the splicing efficiency to 38%, whereas further deletions completely abolished splicing (mutants 2469, 2457 and 2436; Figure 4). To map the regulatory element involved, we made internal 25 bp deletions (Figure 4), and the results showed that the sequence deleted in mutant Δ2457–2482 was sufficient to abolish splicing. This region contains a 9 bp purine-rich GAGAGAAGA sequence, and substitution of the four adenines into pyrimidines induced nearly complete intron retention (ESEmut, Figure 4).Figure 4.

Bottom Line: Moreover, small interfering-mediated silencing of Drosha and/or DGCR8 improves splicing efficiency and abolishes miR-34b production.Thus, the processing of this 3' SO miRNA is regulated in an antagonistic manner by the Microprocessor and the spliceosome owing to competition between these two machineries for the nascent transcript.We propose that this novel mechanism is commonly used to regulate the relative amount of SO miRNA and messenger RNA produced from primary transcripts.

View Article: PubMed Central - PubMed

Affiliation: Human Molecular Genetics, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149, Trieste, Italy.

ABSTRACT
We have explored the functional relationships between spliceosome and Microprocessor complex activities in a novel class of microRNAs (miRNAs), named Splice site Overlapping (SO) miRNAs, whose pri-miRNA hairpins overlap splice sites. We focused on the evolutionarily conserved SO miR-34b, and we identified two indispensable elements for recognition of its 3' splice site: a branch point located in the hairpin and a downstream purine-rich exonic splicing enhancer. In minigene systems, splicing inhibition owing to exonic splicing enhancer deletion or AG 3'ss mutation increases miR-34b levels. Moreover, small interfering-mediated silencing of Drosha and/or DGCR8 improves splicing efficiency and abolishes miR-34b production. Thus, the processing of this 3' SO miRNA is regulated in an antagonistic manner by the Microprocessor and the spliceosome owing to competition between these two machineries for the nascent transcript. We propose that this novel mechanism is commonly used to regulate the relative amount of SO miRNA and messenger RNA produced from primary transcripts.

Show MeSH