Limits...
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.

Show MeSH
Splicing of SO pri-miR-34b affects miRNA biosynthesis. Northern blot analysis of miR-34b and U6 snRNA. The analysis was performed on the AG dinucleotide mutants, on the 5′ss mutant described in Figure 3 (3′ss mut 1, 3′ss mut 2 and 5′ss mut) and on the mutant of the ESE shown in Figure 4 (ESE mut). Histograms show the fold increase of mature miR-34b normalized to U6. The abundance of miR-34b in the wild-type construct is set to 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3794580&req=5

gkt614-F5: Splicing of SO pri-miR-34b affects miRNA biosynthesis. Northern blot analysis of miR-34b and U6 snRNA. The analysis was performed on the AG dinucleotide mutants, on the 5′ss mutant described in Figure 3 (3′ss mut 1, 3′ss mut 2 and 5′ss mut) and on the mutant of the ESE shown in Figure 4 (ESE mut). Histograms show the fold increase of mature miR-34b normalized to U6. The abundance of miR-34b in the wild-type construct is set to 1.

Mentions: To explore the effect of splicing on miRNA biosynthesis, we analysed the miR-34b derived from processing of the pcDNA3pY7 miR-34b minigenes. We evaluated the wild-type construct, 3′ss mut 1 and 3′ss mut 2 that directly affect the AG dinucleotide (Figure 3a), the 5′ss mutant (Figure 3a) and the ESEmut minigene (Figure 4). In comparison with the wild-type, the amount of mature miR-34b was significantly increased by the mutants that affect 3′ss splicing efficiency (Figure 5). In particular, 3′ss mut 1, which reduced the splicing efficiency to ∼50%, was associated with a ∼1.6-fold increase in miR-34b (Figure 5), whereas the 3′ss mut 2 and the ESEmut, which completely abolished splicing, produced ∼4-fold more miR-34b (Figure 5). On the other hand, splicing inhibition caused by mutation of the 5′ss only slightly reduced the amount of miR-34b (Figure 5). This decrease can be due to the facilitating effect of U1snRNP on Drosha processing, as recently suggested (14). In northern blot analysis, we did not observe any band corresponding to the pre-miRNA intermediate derived from transfection of normal or mutant minigenes. This may indicate that processing of the pre-miRNA by Dicer is efficient and is not a rate-limiting step for its maturation. In addition, we can exclude that the mutants affect miRNA abundance through Dicer-dependent pre-miRNA processing.Figure 5.


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 pri-miR-34b affects miRNA biosynthesis. Northern blot analysis of miR-34b and U6 snRNA. The analysis was performed on the AG dinucleotide mutants, on the 5′ss mutant described in Figure 3 (3′ss mut 1, 3′ss mut 2 and 5′ss mut) and on the mutant of the ESE shown in Figure 4 (ESE mut). Histograms show the fold increase of mature miR-34b normalized to U6. The abundance of miR-34b in the wild-type construct is set to 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt614-F5: Splicing of SO pri-miR-34b affects miRNA biosynthesis. Northern blot analysis of miR-34b and U6 snRNA. The analysis was performed on the AG dinucleotide mutants, on the 5′ss mutant described in Figure 3 (3′ss mut 1, 3′ss mut 2 and 5′ss mut) and on the mutant of the ESE shown in Figure 4 (ESE mut). Histograms show the fold increase of mature miR-34b normalized to U6. The abundance of miR-34b in the wild-type construct is set to 1.
Mentions: To explore the effect of splicing on miRNA biosynthesis, we analysed the miR-34b derived from processing of the pcDNA3pY7 miR-34b minigenes. We evaluated the wild-type construct, 3′ss mut 1 and 3′ss mut 2 that directly affect the AG dinucleotide (Figure 3a), the 5′ss mutant (Figure 3a) and the ESEmut minigene (Figure 4). In comparison with the wild-type, the amount of mature miR-34b was significantly increased by the mutants that affect 3′ss splicing efficiency (Figure 5). In particular, 3′ss mut 1, which reduced the splicing efficiency to ∼50%, was associated with a ∼1.6-fold increase in miR-34b (Figure 5), whereas the 3′ss mut 2 and the ESEmut, which completely abolished splicing, produced ∼4-fold more miR-34b (Figure 5). On the other hand, splicing inhibition caused by mutation of the 5′ss only slightly reduced the amount of miR-34b (Figure 5). This decrease can be due to the facilitating effect of U1snRNP on Drosha processing, as recently suggested (14). In northern blot analysis, we did not observe any band corresponding to the pre-miRNA intermediate derived from transfection of normal or mutant minigenes. This may indicate that processing of the pre-miRNA by Dicer is efficient and is not a rate-limiting step for its maturation. In addition, we can exclude that the mutants affect miRNA abundance through Dicer-dependent pre-miRNA processing.Figure 5.

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