Limits...
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

Let-7a pri-miRNA processing is functionally coupled to RNAP II transcription.(a,b) CMV DNA template encoding let-7a pri-miRNA (a) or the corresponding T7 let-7a pri-miRNA (b) was incubated in nuclear extract in the txn/pri-miRNA processing system for the indicated times. Markers (in base pairs) and RNA species are indicated. (c) RNAs were detected by phosphorimager and quantified using Quantity one software. The ratios of pre-let-7a signal at each point (lanes 2–5) to pri-let-7a signal at the start of the reaction (lane 1) were calculated and mean ± S.D. of three biological replicates are shown. The difference in pre/pri ratio between the two groups at each time point was analyzed by Student’s t-test.(d) CMV DNA template encoding let-7a pri-miRNA (lanes 1–4) or naked CMV let-7a pri-miRNA (lanes 5–8) was incubated in nuclear extract under txn/pri-miRNA reaction conditions for the indicated times. Markers (in base pairs) and RNA species are indicated. Ori marks the gel origin. (e) Same as c except that the data in panel d were quantitated. Mean ± S.D. of three biological replicates are shown, and the difference in pre/pri ratio between the two groups was examined using Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493704&req=5

f3: Let-7a pri-miRNA processing is functionally coupled to RNAP II transcription.(a,b) CMV DNA template encoding let-7a pri-miRNA (a) or the corresponding T7 let-7a pri-miRNA (b) was incubated in nuclear extract in the txn/pri-miRNA processing system for the indicated times. Markers (in base pairs) and RNA species are indicated. (c) RNAs were detected by phosphorimager and quantified using Quantity one software. The ratios of pre-let-7a signal at each point (lanes 2–5) to pri-let-7a signal at the start of the reaction (lane 1) were calculated and mean ± S.D. of three biological replicates are shown. The difference in pre/pri ratio between the two groups at each time point was analyzed by Student’s t-test.(d) CMV DNA template encoding let-7a pri-miRNA (lanes 1–4) or naked CMV let-7a pri-miRNA (lanes 5–8) was incubated in nuclear extract under txn/pri-miRNA reaction conditions for the indicated times. Markers (in base pairs) and RNA species are indicated. Ori marks the gel origin. (e) Same as c except that the data in panel d were quantitated. Mean ± S.D. of three biological replicates are shown, and the difference in pre/pri ratio between the two groups was examined using Student’s t-test.

Mentions: We next investigated the efficiency and kinetics of pri-miRNA processing in our txn/pri-miRNA system versus processing of naked pri-miRNAs. To do this, we first carried out a time course comparing txn/pri-miRNA processing using CMV pri-let-7a DNA versus naked T7 pri-let-7a (Fig. 3a–c). This analysis revealed that processing of the RNAP II-generated transcript occurred as early as 5 min of incubation and continued to increase (Fig. 3a). In marked contrast, processing of naked T7 pri-let-7a incubated under the same conditions did not even begin until the 15 min time point (Fig. 3b) and the yields of pre-let-7a were significantly lower (Fig. 3c, p < 0.001, student’s t-test). We conclude that processing of pri-let-7A miRNA in the txn/pri-miRNA system occurs with faster kinetics and efficiency than that of naked T7 pri-miRNA transcripts under the same conditions.


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

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

Let-7a pri-miRNA processing is functionally coupled to RNAP II transcription.(a,b) CMV DNA template encoding let-7a pri-miRNA (a) or the corresponding T7 let-7a pri-miRNA (b) was incubated in nuclear extract in the txn/pri-miRNA processing system for the indicated times. Markers (in base pairs) and RNA species are indicated. (c) RNAs were detected by phosphorimager and quantified using Quantity one software. The ratios of pre-let-7a signal at each point (lanes 2–5) to pri-let-7a signal at the start of the reaction (lane 1) were calculated and mean ± S.D. of three biological replicates are shown. The difference in pre/pri ratio between the two groups at each time point was analyzed by Student’s t-test.(d) CMV DNA template encoding let-7a pri-miRNA (lanes 1–4) or naked CMV let-7a pri-miRNA (lanes 5–8) was incubated in nuclear extract under txn/pri-miRNA reaction conditions for the indicated times. Markers (in base pairs) and RNA species are indicated. Ori marks the gel origin. (e) Same as c except that the data in panel d were quantitated. Mean ± S.D. of three biological replicates are shown, and the difference in pre/pri ratio between the two groups was examined using Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Let-7a pri-miRNA processing is functionally coupled to RNAP II transcription.(a,b) CMV DNA template encoding let-7a pri-miRNA (a) or the corresponding T7 let-7a pri-miRNA (b) was incubated in nuclear extract in the txn/pri-miRNA processing system for the indicated times. Markers (in base pairs) and RNA species are indicated. (c) RNAs were detected by phosphorimager and quantified using Quantity one software. The ratios of pre-let-7a signal at each point (lanes 2–5) to pri-let-7a signal at the start of the reaction (lane 1) were calculated and mean ± S.D. of three biological replicates are shown. The difference in pre/pri ratio between the two groups at each time point was analyzed by Student’s t-test.(d) CMV DNA template encoding let-7a pri-miRNA (lanes 1–4) or naked CMV let-7a pri-miRNA (lanes 5–8) was incubated in nuclear extract under txn/pri-miRNA reaction conditions for the indicated times. Markers (in base pairs) and RNA species are indicated. Ori marks the gel origin. (e) Same as c except that the data in panel d were quantitated. Mean ± S.D. of three biological replicates are shown, and the difference in pre/pri ratio between the two groups was examined using Student’s t-test.
Mentions: We next investigated the efficiency and kinetics of pri-miRNA processing in our txn/pri-miRNA system versus processing of naked pri-miRNAs. To do this, we first carried out a time course comparing txn/pri-miRNA processing using CMV pri-let-7a DNA versus naked T7 pri-let-7a (Fig. 3a–c). This analysis revealed that processing of the RNAP II-generated transcript occurred as early as 5 min of incubation and continued to increase (Fig. 3a). In marked contrast, processing of naked T7 pri-let-7a incubated under the same conditions did not even begin until the 15 min time point (Fig. 3b) and the yields of pre-let-7a were significantly lower (Fig. 3c, p < 0.001, student’s t-test). We conclude that processing of pri-let-7A miRNA in the txn/pri-miRNA system occurs with faster kinetics and efficiency than that of naked T7 pri-miRNA transcripts under the same conditions.

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