BMC Mol Biol. 2011 Feb 9;12:6. doi: 10.1186/1471-2199-12-6.

Two-step cleavage of hairpin RNA with 5' overhangs by human DICER.

Ando Y, Maida Y, Morinaga A, Burroughs AM, Kimura R, Chiba J, Suzuki H, Masutomi K, Hayashizaki Y.

Source

RIKEN Omics Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.

Abstract

BACKGROUND:

DICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs) and long double-stranded RNAs, generating microRNA (miRNA) duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts) in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown.

RESULTS:

In this study, we show that human recombinant DICER protein (rDICER) processes a hairpin RNA with 5' overhangs in vitro and generates an intermediate duplex with a 29 nt-5' strand and a 23 nt-3' strand, which was eventually cleaved into a canonical miRNA duplex via a two-step cleavage. The previously identified endogenous pre-miRNA with 5' overhangs, pre-mmu-mir-1982 RNA, is also determined to be a substrate of rDICER through the same two-step cleavage.

CONCLUSIONS:

The two-step cleavage of a hairpin RNA with 5' overhangs shows that DICER releases double-stranded RNAs after the first cleavage and binds them again in the inverse direction for a second cleavage. These findings have implications for how DICER may be able to interact with or process differing precursor structures.

PMID:: 21306637; [PubMed - indexed for MEDLINE]
PMCID:: PMC3048551

Free PMC Article

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Figure 1

Characterization of recombinant DICER protein. Purified rDICER (3 μg) was loaded onto a NuPAGE 4-12% Bis-Tris gel. Proteins were analyzed by Coomassie Brilliant Blue (CBB) staining (A) and Western blotting with anti-DICER antibody (B). (C) Western blotting with anti-DICER, anti-FLAG, anti-AGO2 and anti-TRBP antibodies. 30 μg of 293T cell lysate (lane 1) and 3 μg of rDICER (lane 2) were loaded.

Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

BMC Mol Biol. 2011;12:6-6.

Figure 2

Processing of the precursor miRNA by recombinant DICER protein. (A) Precursor miRNA (pre-mir-21 RNA) and probe used in this study. The 5' end nucleotide of pre-mir-21 RNA was modified (U to G) from human pre-hsa-mir-21 RNA sequence registered in miRBase 14.0 [46-48] in order to facilitate the in vitro transcription reaction. The secondary structure was predicted using the CentroidFold program [49]. The solid line shows the position of probe-1. (B) Detection of the rDICER-processed products by Northern blotting. Pre-mir-21 RNAs were incubated with rDICER in vitro for the indicated time points (0, 10, 20, 30, 40, 50, 60, 75, 90, 105 and 120 min). The RNAs processed by rDICER were detected using probe-1 by Northern blotting. The gray arrow shows the band of pre-mir-21 RNA and the black arrow shows the band of miRNA processed from the 5' strand of pre-mir-21 RNA. The asterisk shows the nicked product (~37 nt) similar to a previous report [29].

Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

BMC Mol Biol. 2011;12:6-6.

Figure 3

Processing of the hairpin RNA with 5' overhangs by recombinant DICER protein. (A) Hairpin RNA with 5' overhangs (RNA-I) and probes used in this study. RNA-I was a hairpin RNA with 5' overhangs based on the "pre-mir-21 RNA" sequence. The secondary structure was predicted using the CentroidFold program [49]. The solid line shows the position of probe-1, the dashed line shows the position of probe-2 and the dotted line shows the position of probe-3. (B-D) Time-course analysis of the processing of RNA-I by the rDICER protein. RNA-I RNAs were incubated with rDICER in vitro for the indicated time points (0, 10, 20, 30, 40, 50, 60, 75, 90, 105 and 120 min). The RNAs processed by rDICER were detected using probe-1, probe-2 and probe-3 (B-D, respectively) by Northern blotting. The gray arrow shows the band of unprocessed RNA-I and the black arrow shows the bands of small RNA processed from the 5' strand, 3' strand and loop region of RNA-I respectively. M: decade marker. (E) The processing of RNA-I by the rDICER protein at a longer incubation time. RNA-I RNAs were incubated with rDICER for 30 min and 16 hours. The RNAs processed from the 5' strand of RNA-I were detected using probe-1 by Northern blotting.

Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

BMC Mol Biol. 2011;12:6-6.

Figure 4

Two-step processing of the 5'-end labelled RNA-I by recombinant DICER protein. (A) In vitro processing of the 5'-end labelled RNA-I by rDICER protein. 5'-end labelled RNA-I RNAs were incubated without rDICER for 120 min (lane 1) and with rDICER for the following time points (0, 10, 20, 30, 40, 50, 60 and 120 min; lane 2-9 respectively). The processing reaction was faster than the results of Figure 3B because the amount of RNA substrate in this reaction mixture was less. The RNA products less than 10 nt look stacked at the end of the gel because of the difficulty in separating efficiently, even at 7.5 M urea denaturing 20% polyacrylamide sequence gel. This experiment was repeated and replicated consistently. M: decade marker. (B) Sequences of band 1 and 2 in Figure 3B identified from Figure 4A and Table 1. Sequences highlighted in gray are 29-nt (band 1) and 23-nt RNA (band 2) from the 5' strand, respectively.

Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

BMC Mol Biol. 2011;12:6-6.

Figure 5

Model of the two-step processing of hairpin RNA with 5' overhangs (RNA-I) by DICER protein. rDICER processes hairpin RNA with 5' overhangs (RNA-I) to dsRNA with 29 nt-5' strand and 23 nt-3' strand after the first cleavage reaction and releases once from the binding site. Then, the dsRNA is bound in the inverse direction with the same or different rDICER molecule and is measured after the anchoring 3' end of the 29-nt strand to generate dsRNA with 23 nt cleaved from the 29-nt strand and 22 nt cleaved from the 23-nt strand. "PAZ" domain of rDICER colored purple; "RIIIa" and "RIIIb" domain of rDICER colored blue. Lightning marks indicate the cleavage sites in the RNA.

Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

BMC Mol Biol. 2011;12:6-6.

Figure 6

Processing of pre-mmu-mir-1982 RNA by recombinant DICER protein. (A) Pre-mmu-mir-1982 RNA and probes used in this study. The secondary structure was predicted using the CentroidFold program [49]. The solid line shows the position of probe-4 and the dashed line shows the position of probe-5. (B-C) Time-course analysis of the processing of pre-mmu-mir-1982 RNA by the rDICER protein. pre-mmu-mir-1982 RNAs were incubated with rDICER in vitro for the indicated time points (0, 10, 20, 30, 40, 50, 60, 75, 90, 105 and 120 min). The RNAs processed by rDICER were detected using probe-4 (B), probe-5 (C) by Northern blotting. The gray arrow shows the band of unprocessed RNA and the black arrow shows the bands of small RNA processed from the 5' strand and 3' strand of pre-mmu-mir-1982 RNA respectively. M: decade marker.

Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

BMC Mol Biol. 2011;12:6-6.

Figure 7

Two-step processing of the 5'-end labelled pre-mmu-mir-1982 RNA by recombinant DICER protein. (A) In vitro processing of the pre-mmu-mir-1982 RNA by the rDICER protein at a longer incubation time. 5' labelled pre-mmu-mir-1982 RNAs were incubated with rDICER for 0, 2 and 16 hours. The gray arrow shows the band of unprocessed RNA and the black arrow shows the bands of small RNA processed from pre-mmu-mir-1982 RNA. AH: the alkaline hydrolysis ladder of pre-mmu-mir-1982 RNA. The size of each band was determined by the AH ladder. (B-C) The signal intensities were quantified from the 12 nt (B) and 35 nt (C) bands in Figure 7A. These plots show average values bracketed by s.e.m. error bars; calculated from two independent experiments. Background refers to the signal intensity of the same sized band in the AH lane. The p-value was calculated using a simple t-test for each time point (2 hrs and 16 hrs) relative to the background. Significant differences (p < 0.05) in signal intensities are denoted with an asterisk. The significant calculated p-values are as follows: the 12-nt band at 16 hours, p = 0.017; the 35-nt band at 2 hours, p = 0.0073; and the 35-nt band at 16 hours, p = 0.024.

Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

BMC Mol Biol. 2011;12:6-6.

Publication Types, MeSH Terms, Substances

Publication Types

Research Support, Non-U.S. Gov't

MeSH Terms

DEAD-box RNA Helicases/chemistry
DEAD-box RNA Helicases/genetics
DEAD-box RNA Helicases/metabolism*
Humans
MicroRNAs/metabolism
RNA, Double-Stranded/chemistry
RNA, Double-Stranded/metabolism*
Recombinant Proteins/chemistry
Recombinant Proteins/genetics
Recombinant Proteins/metabolism
Ribonuclease III/chemistry
Ribonuclease III/genetics
Ribonuclease III/metabolism*

Substances

MicroRNAs
RNA, Double-Stranded
Recombinant Proteins
DICER1 protein, human
Ribonuclease III
DEAD-box RNA Helicases

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Two-step cleavage of hairpin RNA with 5' overhangs by human DICER.

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Abstract

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RESULTS:

CONCLUSIONS:

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