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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.


RNAP II txn/pri-miRNA processing system in vitro.(a) Structure of the CMV DNA template encoding let-7a pri-miRNA used for txn/pri-miRNA processing. The sizes of 5′ flanking region, 3’ flanking region, and pre-let-7a are indicated. The thick line indicates the natural pri-miRNA sequences and the thin lines indicate the vector sequences. CMV-Δpre-let-7a is a mutant lacking the 72 nt pre-let-7a hairpin. (b) RNAP II txn/pri-miRNA processing was carried out for the indicated times using the CMV DNA template encoding let-7a pri-miRNA (lanes 1, 2), the CMV DNA template encoding let-7a Δpre-miRNA DNA template (lanes 3, 4) or no DNA template (lanes 5, 6). The asterisk indicates the 3′ flanking region, which lacks a cap and is therefore largely degraded during the reaction (lane 2). (c) RNAP II txn/pri-miRNA processing was carried using different amounts of MgCl2 as indicated and the CMV DNA template encoding let-7a pri-miRNA. Size markers (in base pairs) and RNA species are indicated. Ori indicates the gel origin. RNAs were detected by phosphorimager and quantified using Quantity One software. Mean ± S.D. of three biological replicates are shown.
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f1: RNAP II txn/pri-miRNA processing system in vitro.(a) Structure of the CMV DNA template encoding let-7a pri-miRNA used for txn/pri-miRNA processing. The sizes of 5′ flanking region, 3’ flanking region, and pre-let-7a are indicated. The thick line indicates the natural pri-miRNA sequences and the thin lines indicate the vector sequences. CMV-Δpre-let-7a is a mutant lacking the 72 nt pre-let-7a hairpin. (b) RNAP II txn/pri-miRNA processing was carried out for the indicated times using the CMV DNA template encoding let-7a pri-miRNA (lanes 1, 2), the CMV DNA template encoding let-7a Δpre-miRNA DNA template (lanes 3, 4) or no DNA template (lanes 5, 6). The asterisk indicates the 3′ flanking region, which lacks a cap and is therefore largely degraded during the reaction (lane 2). (c) RNAP II txn/pri-miRNA processing was carried using different amounts of MgCl2 as indicated and the CMV DNA template encoding let-7a pri-miRNA. Size markers (in base pairs) and RNA species are indicated. Ori indicates the gel origin. RNAs were detected by phosphorimager and quantified using Quantity One software. Mean ± S.D. of three biological replicates are shown.

Mentions: In previous in vitro studies, pri-miRNA processing was carried out in total cell lysates or nuclear extracts using naked pri-miRNA transcripts212223. Although pri-miRNAs were processed, the efficiency was low. Consistent with these studies, we found a low level of processing of naked T7 pri-let-7a in HeLa cell nuclear extract (Supplementary Fig. S1a,b). In previous work, we found that pre-mRNA splicing was potently enhanced when nascent pre-mRNA was transcribed by RNAP II14. As miRNAs are also synthesized by RNAP II, we asked whether transcription by RNAP II affects pri-miRNA processing in vitro. To do this, we constructed a DNA template driven by the CMV promoter and encoding let-7a pri-miRNA (Supplementary Fig. S2a). For comparison, we used a naked T7 transcript encoding let-7a pri-miRNA. We first tested our RNAP II transcription/splicing (txn/splicing) conditions for pri-miRNA processing. This reaction mixture contains nuclear extract, 32P-UTP, 3.2 mM MgCl2, ATP, and an ATP regenerating system (see Methods)14. We carried out these reactions in both the presence and absence of the crowding agent PVA, which is known to enhance gene expression steps in vitro1533 (Supplementary Fig. S2b,c). In addition, we tested whether formation of an RNAP II pre-initiation complex (PIC)1833 stimulated pri-mRNA processing (Supplementary Fig. S2d,e). Both U6 snRNA and tRNA are known to be labeled by 32P-UTP in our coupled txn/splicing system1434, and both of these RNA species were detected under all conditions we tested (Supplementary Fig. S2b–d). When we used PIC conditions in the presence of PVA, we observed a large increase of RNAP II transcription, and faint bands that could correspond to pri-miRNA processing products were also detected (Supplementary Fig. S2d). Thus, we further optimized this system. To do this, we carried out transcription for 5 min using PIC conditions with PVA, diluted this reaction four fold into fresh nuclear extract, followed by continued incubation for 0 or 10 min at 37 °C (Fig. 1b, lanes 1 and 2). Strikingly, the combination of PIC formation in the presence of PVA and addition of fresh nuclear extract led to a potent enhancement of let-7a pri-miRNA processing. At the 10-min time point, bands of the expected sizes for the 5′ and 3′ flanks (263, 253 nts, respectively, Fig. 1a) were detected (Fig. 1b, lane 2). The putative 3′ flank (designated by *) is largely degraded, most likely because it lacks a 5′ cap. A band of the expected size for the pre-let-7a miRNA was also observed (72 nts, Fig. 2b, lane 2; and see below for identification of processing products). The putative processing is specific as it was not observed when we used CMV-Δpre-let-7a, a construct in which the 72 nt stem-loop of the pre-miRNA was deleted (see schematic in Fig. 1a and Fig. 1b, lanes 3 and 4) or when DNA was omitted from the reaction mixture (Fig. 1b, lanes 5 and 6). In contrast to the efficient processing observed with this system, there was no significant effect on processing of naked T7 pri-miRNA let-7a under the same conditions (see below). We also determined the optimal MgCl2 concentration for the txn/pri-miRNA processing system. Consistent with the naked T7 pri-miRNA processing system, we found that the optimal concentration is 6.4 mM, but processing was still efficient at all concentrations tested within the range of 1.6 mM to 12.8 mM (Fig. 1c, lanes 2–5).


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

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

RNAP II txn/pri-miRNA processing system in vitro.(a) Structure of the CMV DNA template encoding let-7a pri-miRNA used for txn/pri-miRNA processing. The sizes of 5′ flanking region, 3’ flanking region, and pre-let-7a are indicated. The thick line indicates the natural pri-miRNA sequences and the thin lines indicate the vector sequences. CMV-Δpre-let-7a is a mutant lacking the 72 nt pre-let-7a hairpin. (b) RNAP II txn/pri-miRNA processing was carried out for the indicated times using the CMV DNA template encoding let-7a pri-miRNA (lanes 1, 2), the CMV DNA template encoding let-7a Δpre-miRNA DNA template (lanes 3, 4) or no DNA template (lanes 5, 6). The asterisk indicates the 3′ flanking region, which lacks a cap and is therefore largely degraded during the reaction (lane 2). (c) RNAP II txn/pri-miRNA processing was carried using different amounts of MgCl2 as indicated and the CMV DNA template encoding let-7a pri-miRNA. Size markers (in base pairs) and RNA species are indicated. Ori indicates the gel origin. RNAs were detected by phosphorimager and quantified using Quantity One software. Mean ± S.D. of three biological replicates are shown.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493704&req=5

f1: RNAP II txn/pri-miRNA processing system in vitro.(a) Structure of the CMV DNA template encoding let-7a pri-miRNA used for txn/pri-miRNA processing. The sizes of 5′ flanking region, 3’ flanking region, and pre-let-7a are indicated. The thick line indicates the natural pri-miRNA sequences and the thin lines indicate the vector sequences. CMV-Δpre-let-7a is a mutant lacking the 72 nt pre-let-7a hairpin. (b) RNAP II txn/pri-miRNA processing was carried out for the indicated times using the CMV DNA template encoding let-7a pri-miRNA (lanes 1, 2), the CMV DNA template encoding let-7a Δpre-miRNA DNA template (lanes 3, 4) or no DNA template (lanes 5, 6). The asterisk indicates the 3′ flanking region, which lacks a cap and is therefore largely degraded during the reaction (lane 2). (c) RNAP II txn/pri-miRNA processing was carried using different amounts of MgCl2 as indicated and the CMV DNA template encoding let-7a pri-miRNA. Size markers (in base pairs) and RNA species are indicated. Ori indicates the gel origin. RNAs were detected by phosphorimager and quantified using Quantity One software. Mean ± S.D. of three biological replicates are shown.
Mentions: In previous in vitro studies, pri-miRNA processing was carried out in total cell lysates or nuclear extracts using naked pri-miRNA transcripts212223. Although pri-miRNAs were processed, the efficiency was low. Consistent with these studies, we found a low level of processing of naked T7 pri-let-7a in HeLa cell nuclear extract (Supplementary Fig. S1a,b). In previous work, we found that pre-mRNA splicing was potently enhanced when nascent pre-mRNA was transcribed by RNAP II14. As miRNAs are also synthesized by RNAP II, we asked whether transcription by RNAP II affects pri-miRNA processing in vitro. To do this, we constructed a DNA template driven by the CMV promoter and encoding let-7a pri-miRNA (Supplementary Fig. S2a). For comparison, we used a naked T7 transcript encoding let-7a pri-miRNA. We first tested our RNAP II transcription/splicing (txn/splicing) conditions for pri-miRNA processing. This reaction mixture contains nuclear extract, 32P-UTP, 3.2 mM MgCl2, ATP, and an ATP regenerating system (see Methods)14. We carried out these reactions in both the presence and absence of the crowding agent PVA, which is known to enhance gene expression steps in vitro1533 (Supplementary Fig. S2b,c). In addition, we tested whether formation of an RNAP II pre-initiation complex (PIC)1833 stimulated pri-mRNA processing (Supplementary Fig. S2d,e). Both U6 snRNA and tRNA are known to be labeled by 32P-UTP in our coupled txn/splicing system1434, and both of these RNA species were detected under all conditions we tested (Supplementary Fig. S2b–d). When we used PIC conditions in the presence of PVA, we observed a large increase of RNAP II transcription, and faint bands that could correspond to pri-miRNA processing products were also detected (Supplementary Fig. S2d). Thus, we further optimized this system. To do this, we carried out transcription for 5 min using PIC conditions with PVA, diluted this reaction four fold into fresh nuclear extract, followed by continued incubation for 0 or 10 min at 37 °C (Fig. 1b, lanes 1 and 2). Strikingly, the combination of PIC formation in the presence of PVA and addition of fresh nuclear extract led to a potent enhancement of let-7a pri-miRNA processing. At the 10-min time point, bands of the expected sizes for the 5′ and 3′ flanks (263, 253 nts, respectively, Fig. 1a) were detected (Fig. 1b, lane 2). The putative 3′ flank (designated by *) is largely degraded, most likely because it lacks a 5′ cap. A band of the expected size for the pre-let-7a miRNA was also observed (72 nts, Fig. 2b, lane 2; and see below for identification of processing products). The putative processing is specific as it was not observed when we used CMV-Δpre-let-7a, a construct in which the 72 nt stem-loop of the pre-miRNA was deleted (see schematic in Fig. 1a and Fig. 1b, lanes 3 and 4) or when DNA was omitted from the reaction mixture (Fig. 1b, lanes 5 and 6). In contrast to the efficient processing observed with this system, there was no significant effect on processing of naked T7 pri-miRNA let-7a under the same conditions (see below). We also determined the optimal MgCl2 concentration for the txn/pri-miRNA processing system. Consistent with the naked T7 pri-miRNA processing system, we found that the optimal concentration is 6.4 mM, but processing was still efficient at all concentrations tested within the range of 1.6 mM to 12.8 mM (Fig. 1c, lanes 2–5).

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.