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Primary microRNA processing is functionally coupled to RNAP II transcription in vitro.

Yin S, Yu Y, Reed R - Sci Rep (2015)

Bottom Line: We show that both the kinetics and efficiency of pri-miRNA processing are dramatically enhanced in this system compared to that of the corresponding naked pri-miRNA.We also show that nascent pri-miRNA is efficiently processed before it is released from the DNA template.Together, our work directly demonstrates that transcription and pri-miRNA processing are functionally coupled and establishes the first in vivo model systems for this functional coupling and for co-transcriptional processing.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Ave. Boston MA 02115.

ABSTRACT
Previous studies in vivo reported that processing of primary microRNA (pri-miRNA) is coupled to transcription by RNA polymerase II (RNAP II) and can occur co-transcriptionally. Here we have established a robust in vivo system in which pri-miRNA is transcribed by RNAP II and processed to pre-miRNA in HeLa cell nuclear extracts. We show that both the kinetics and efficiency of pri-miRNA processing are dramatically enhanced in this system compared to that of the corresponding naked pri-miRNA. Moreover, this enhancement is general as it occurs with multiple pri-miRNAs. We also show that nascent pri-miRNA is efficiently processed before it is released from the DNA template. Together, our work directly demonstrates that transcription and pri-miRNA processing are functionally coupled and establishes the first in vivo model systems for this functional coupling and for co-transcriptional processing.

No MeSH data available.


Related in: MedlinePlus

Pri-let-7a miRNA processing occurs on an immobilized DNA template.(a) Schematic of CMV pri-let-7a DNA template bound to streptavidin magnetic beads via biotin at the 3′ end. (b) CMV pri-let-7a DNA templates containing biotin or no biotin were incubated with streptavidin magnetic beads. DNA templates in bound (bd) and supernatant (sp) fractions were analyzed on a 1% agarose gel by staining with ethidium bromide. (c) Biotinylated CMV pri-let-7a DNA template was 3′ end-labeled, bound to streptavidin magnetic beads and incubated in nuclear extract at 30o for 30 min. Bound and supernatant fractions were analyzed on an 8% denaturing polyacrylamide gel. (d) CMV pri-let-7a DNA templates containing biotin or no biotin were incubated in nuclear extracts under txn/processing conditions for the indicated times. Total RNA was isolated and fractionated on an 8% denaturing polyacrylamide gel. (e) Streptavidin magnetic beads were incubated with CMV pri-let-7a DNA templates containing biotin or no biotin. After washing, beads with or without immobilized DNA templates were incubated in nuclear extracts under txn/processing conditions for the times indicated. Total RNA from each sample was isolated and fractionated on an 8% denaturing polyacrylamide gel. (f) Immobilized CMV pri-let-7a DNA template was incubated under txn/processing conditions for the times indicated. The bound and supernatant fractions were separated, and the bound fraction was washed. Total RNA was then isolated from the bound and supernatant fractions and run on an 8% denaturing polyacrylamide gel.
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f4: Pri-let-7a miRNA processing occurs on an immobilized DNA template.(a) Schematic of CMV pri-let-7a DNA template bound to streptavidin magnetic beads via biotin at the 3′ end. (b) CMV pri-let-7a DNA templates containing biotin or no biotin were incubated with streptavidin magnetic beads. DNA templates in bound (bd) and supernatant (sp) fractions were analyzed on a 1% agarose gel by staining with ethidium bromide. (c) Biotinylated CMV pri-let-7a DNA template was 3′ end-labeled, bound to streptavidin magnetic beads and incubated in nuclear extract at 30o for 30 min. Bound and supernatant fractions were analyzed on an 8% denaturing polyacrylamide gel. (d) CMV pri-let-7a DNA templates containing biotin or no biotin were incubated in nuclear extracts under txn/processing conditions for the indicated times. Total RNA was isolated and fractionated on an 8% denaturing polyacrylamide gel. (e) Streptavidin magnetic beads were incubated with CMV pri-let-7a DNA templates containing biotin or no biotin. After washing, beads with or without immobilized DNA templates were incubated in nuclear extracts under txn/processing conditions for the times indicated. Total RNA from each sample was isolated and fractionated on an 8% denaturing polyacrylamide gel. (f) Immobilized CMV pri-let-7a DNA template was incubated under txn/processing conditions for the times indicated. The bound and supernatant fractions were separated, and the bound fraction was washed. Total RNA was then isolated from the bound and supernatant fractions and run on an 8% denaturing polyacrylamide gel.

Mentions: In previous work, we found that pre-mRNA splicing could occur while the CMV-DNA was immobilized on beads14. Thus, we investigated whether pri-miRNA processing can occur on an immobilized DNA template (see schematic, Fig. 4a). The CMV DNA template encoding let-7a pri-miRNA was amplified using a 3′ primer that was end-labeled with biotin and a CMV let-7a pri-miRNA DNA template lacking biotin was used as a negative control. As shown in Fig. 4b, only the biotinylated PCR product (lanes 1–3) but not the non-biotinylated PCR product (lanes 4–6) bound efficiently to the streptavidin beads. Moreover, the biotinylated32, P-labeled CMV-let-7a DNA template did not detach from the beads when they were incubated for 30 min in nuclear extract under coupled txn/pri-miRNA processing conditions, as indicated by the presence of the labeled, biotinylated DNA template only in the bound, but not the supernatant fraction (Fig. 4c).


Primary microRNA processing is functionally coupled to RNAP II transcription in vitro.

Yin S, Yu Y, Reed R - Sci Rep (2015)

Pri-let-7a miRNA processing occurs on an immobilized DNA template.(a) Schematic of CMV pri-let-7a DNA template bound to streptavidin magnetic beads via biotin at the 3′ end. (b) CMV pri-let-7a DNA templates containing biotin or no biotin were incubated with streptavidin magnetic beads. DNA templates in bound (bd) and supernatant (sp) fractions were analyzed on a 1% agarose gel by staining with ethidium bromide. (c) Biotinylated CMV pri-let-7a DNA template was 3′ end-labeled, bound to streptavidin magnetic beads and incubated in nuclear extract at 30o for 30 min. Bound and supernatant fractions were analyzed on an 8% denaturing polyacrylamide gel. (d) CMV pri-let-7a DNA templates containing biotin or no biotin were incubated in nuclear extracts under txn/processing conditions for the indicated times. Total RNA was isolated and fractionated on an 8% denaturing polyacrylamide gel. (e) Streptavidin magnetic beads were incubated with CMV pri-let-7a DNA templates containing biotin or no biotin. After washing, beads with or without immobilized DNA templates were incubated in nuclear extracts under txn/processing conditions for the times indicated. Total RNA from each sample was isolated and fractionated on an 8% denaturing polyacrylamide gel. (f) Immobilized CMV pri-let-7a DNA template was incubated under txn/processing conditions for the times indicated. The bound and supernatant fractions were separated, and the bound fraction was washed. Total RNA was then isolated from the bound and supernatant fractions and run on an 8% denaturing polyacrylamide gel.
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Related In: Results  -  Collection

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f4: Pri-let-7a miRNA processing occurs on an immobilized DNA template.(a) Schematic of CMV pri-let-7a DNA template bound to streptavidin magnetic beads via biotin at the 3′ end. (b) CMV pri-let-7a DNA templates containing biotin or no biotin were incubated with streptavidin magnetic beads. DNA templates in bound (bd) and supernatant (sp) fractions were analyzed on a 1% agarose gel by staining with ethidium bromide. (c) Biotinylated CMV pri-let-7a DNA template was 3′ end-labeled, bound to streptavidin magnetic beads and incubated in nuclear extract at 30o for 30 min. Bound and supernatant fractions were analyzed on an 8% denaturing polyacrylamide gel. (d) CMV pri-let-7a DNA templates containing biotin or no biotin were incubated in nuclear extracts under txn/processing conditions for the indicated times. Total RNA was isolated and fractionated on an 8% denaturing polyacrylamide gel. (e) Streptavidin magnetic beads were incubated with CMV pri-let-7a DNA templates containing biotin or no biotin. After washing, beads with or without immobilized DNA templates were incubated in nuclear extracts under txn/processing conditions for the times indicated. Total RNA from each sample was isolated and fractionated on an 8% denaturing polyacrylamide gel. (f) Immobilized CMV pri-let-7a DNA template was incubated under txn/processing conditions for the times indicated. The bound and supernatant fractions were separated, and the bound fraction was washed. Total RNA was then isolated from the bound and supernatant fractions and run on an 8% denaturing polyacrylamide gel.
Mentions: In previous work, we found that pre-mRNA splicing could occur while the CMV-DNA was immobilized on beads14. Thus, we investigated whether pri-miRNA processing can occur on an immobilized DNA template (see schematic, Fig. 4a). The CMV DNA template encoding let-7a pri-miRNA was amplified using a 3′ primer that was end-labeled with biotin and a CMV let-7a pri-miRNA DNA template lacking biotin was used as a negative control. As shown in Fig. 4b, only the biotinylated PCR product (lanes 1–3) but not the non-biotinylated PCR product (lanes 4–6) bound efficiently to the streptavidin beads. Moreover, the biotinylated32, P-labeled CMV-let-7a DNA template did not detach from the beads when they were incubated for 30 min in nuclear extract under coupled txn/pri-miRNA processing conditions, as indicated by the presence of the labeled, biotinylated DNA template only in the bound, but not the supernatant fraction (Fig. 4c).

Bottom Line: We show that both the kinetics and efficiency of pri-miRNA processing are dramatically enhanced in this system compared to that of the corresponding naked pri-miRNA.We also show that nascent pri-miRNA is efficiently processed before it is released from the DNA template.Together, our work directly demonstrates that transcription and pri-miRNA processing are functionally coupled and establishes the first in vivo model systems for this functional coupling and for co-transcriptional processing.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Ave. Boston MA 02115.

ABSTRACT
Previous studies in vivo reported that processing of primary microRNA (pri-miRNA) is coupled to transcription by RNA polymerase II (RNAP II) and can occur co-transcriptionally. Here we have established a robust in vivo system in which pri-miRNA is transcribed by RNAP II and processed to pre-miRNA in HeLa cell nuclear extracts. We show that both the kinetics and efficiency of pri-miRNA processing are dramatically enhanced in this system compared to that of the corresponding naked pri-miRNA. Moreover, this enhancement is general as it occurs with multiple pri-miRNAs. We also show that nascent pri-miRNA is efficiently processed before it is released from the DNA template. Together, our work directly demonstrates that transcription and pri-miRNA processing are functionally coupled and establishes the first in vivo model systems for this functional coupling and for co-transcriptional processing.

No MeSH data available.


Related in: MedlinePlus