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Autoantigen La regulates miRNA processing from Stem-loop precursors by association with DGCR8 Quan Zheng, Haijie Yang, and Yu-Ren Adam Yuan Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00693 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 1, 2017
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Biochemistry
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Autoantigen La regulates miRNA processing from Stem-loop precursors by
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association with DGCR8
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Quan Zheng,a Hai-Jie Yang,a and Y. Adam Yuan a,b*
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a
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University of Singapore, 14 Science Drive 4, Singapore, 117543, Singapore
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b
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Suzhou Industrial Park, Jiangsu, 215123, China
Department of Biological Sciences and Centre for Bioimaging Sciences, National
National University of Singapore (Suzhou) Research Institute, 377 Lin Quan Street,
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*Address Correspondence to Y. Adam Yuan, Department of Biological Sciences, National
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University of Singapore, 14 Science Drive 4, Singapore, 117543, Singapore. Phone:
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(65)-65162724, Fax: (65)-67792486, Email:
[email protected] 1
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Abstract
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In human, primary microRNA (pri-miRNA) processing starts from precise cleavage of
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the stem loop, which is catalyzed by Drosha-DGCR8 complex. However, the significant
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inconsistencies in the expression levels among primary, precursor and mature miRNAs
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clearly indicate that many other factors may be involved in this regulation. Here, we utilize
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a newly-developed RNA affinity technique to isolate such factors. In this study, a
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tRNA-scaffolded aptamer (tRSA)-based RNA affinity tag, by directly fusing primary let-7
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miRNA to the 3’-end of tRSA, is employed to pull down the protein components
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specifically binding to pri-let-7. We show that La protein binds to pri-let-7 via its La motif
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and significantly promotes the processing efficiency of pri-let-7 in vitro and in cells. In
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addition, we demonstrate that La protein is associated with DGCR8, but not Drosha, in an
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RNA-dependent manner. Interestingly, the RNA binding capacity of La motif is important
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for miRNA processing. Hence, we propose that La protein is an important microprocessor
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component regulating miRNA processing efficiency by association with DGCR8 to
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regulate DGCR8-Drosha complex formation for miRNA processing.
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Biochemistry
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Keywords
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Autoantigen La; microprocessor; microRNA processing; RNA-pull down assay; DGCR8
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Abbreviations
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pri-miRNA, primary microRNA; pre-miRNA, precursor microRNA; tRSA, tRNA-scaffolded
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streptavidin aptamer; RRM, RNA recognition motif; SBM, short basic motif; shLa,
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short-hairpin miRNA knocking-down La expression
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Introduction
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MicroRNAs (miRNAs) belong to a class of noncoding small RNAs and have been
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shown to be integrated into the RISC complex to regulate gene expression through
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repressing translation and/or cleaving mRNAs
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associated with human diseases 4–6, which indicates that the precise control of microRNA
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expression and processing accuracy at different steps are critical for many important
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biological activities in vivo. In human, the primary transcripts of miRNA genes
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(pri-miRNAs) are cleaved to hairpin intermediates (pre-miRNAs) by the nuclear RNase III
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Drosha and two copies of DGCR8 molecules; then further processed to mature miRNAs
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by cytosolic Dicer, another RNaseIII-related enzyme
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RNA-associated proteins are associated with Drosha-DGCR8, the key microprocessor
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components responsible for pri-miRNA processing, the exact functions of these
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RNA-associated proteins in regulating miRNA processing are largely unknown 8. Hence,
1–3
. miRNA dysregulation is often
1,4,5,7
3
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. Notably, although many
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the functional characterization of these microprocessor associated components needs to
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be identified 9,10. In literature, both protein-mediated and RNA-mediated affinity strategies
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are developed to identify and/or map protein-protein, protein-RNA ‘interactome’ via high
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throughput proteomics analysis
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track protein assembly and localization, RNA aptamer-mediated strategies are used to
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track RNA localization in living cells and to isolate RNA-protein complexes by affinity
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chromatographic methods 14.
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11–13
. In contrast to protein-mediated affinity strategies to
MicroRNA processing is an RNA-centered multiple-step maturation process, which is 15,16
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processed by different RNA-protein complexes
. We speculate that specifically
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designed RNA-based affinity strategies could have the possibility to pull down the
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proteins specifically recruited to primary, precursor and mature miRNAs, respectively. To
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this end, we modified the tRNA-scaffolded streptavidin aptamer (tRSA)
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used as an ideal RNA tag to isolate RNA-protein complexes and protein components
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involved in miRNA processing. Here, we described the strategy to fuse pri-let-7 to the
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3’-end of tRSA as the affinity tag to specifically identify novel pri-miRNA binding proteins
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from cell lysates.
17
, which was
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Autoantigen La protein (La/SSB) is known as a nuclear expressed RNA-binding protein
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comprising three RNA-binding domains (the La motif and two RNA recognition motifs).
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The cellular function of La protein has been associated with processing of RNA
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polymerase III transcripts, mRNA stabilization and tRNA recognition through the 3’ UUU
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termini18–20. In this study, among many pre-let-7 binding proteins identified by our 4
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tRSA-based affinity experiments, La protein was discovered to specifically bind to
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pri-let-7 via its La motif and significantly promote the processing efficiency of pri-let-7 in
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vitro and in cells. Moreover, microarray data showed that autoantigen La protein
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regulates processing of a large spectrum of miRNAs by targeting stem-loop miRNA
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precursors, which was validated by miRNA processing assays in vitro. Lastly, in vitro pull
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down assays showed that La protein is associated with DGCR8, but not DROSHA,
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suggesting a novel function of La protein in regulating miRNA processing by affecting
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DGCR8-DROSHA mediated microprocessor assembly.
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Materials and methods
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Plasmid construction
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The sequences of streptavidin aptamer (SA) and tRNA-scaffolded SA tags
17
were
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listed below, which were generated by primer annealing and PCR. The purified DNA
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fragments were digested with Mun I and EcoR I, and ligated into the EcoR I site of
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pcDNA5/FRT
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5’-CAATTGGTCGACCGACCAGAATCATGCAAGTGCGTAAGATAGTCGCGGGCCGG
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GGGCGTATTATGTGCGTCTACATGAATTC-3’;
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5’-CAATTGAAAAAAAAAAAAAGCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCTCG
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ACCAGAATCATGCAAGTGCGTAAGATAGTCGCGGGTCGAGGCCGCGTCCAGGGTT
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CAAGTCCCTGTTCGGGCGCCACTGCAGAAAAAAAAAAAAGAATTC-3’.
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vector
(Invitrogen)
to
construct
pcDNA5/tRSA.
tRNA-scaffolded
SA
SA
(tRSA)
tag:
tag:
To generate pri-let-7a expression plasmid, pri-let-7a was amplified from HEK293 5
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genomic DNA by PCR, digested by BamH I/Not I and cloned into pcDNA5/tRSA. The
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primers for cloning pri-let-7a were shown as following: Forward primer: 5’- CGGGATCC
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AAACTTCATTTTCAACGTAAGTG-3’;
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5’-ATAAGAATGCGGCCGCATCCAGTGTACTTGCTACAG-3’.
Reverse
primer:
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tRSA pull-down assay
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HEK293 cells were transfected with pcDNA5/tRSA and pcDNA5/tRSA-pri-let-7a,
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respectively, using polyethylenimine (PEI) as transfection reagent. In order to avoid
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pri-miRNA being processed in transfected cells, DROSHA expression was knocked down
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by
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pcDNA5/tRSA-pri-let-7a. Cells were harvested 48 h post-transfection by a wash with PBS,
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followed by brief vortex and incubation on ice with lysis buffer (20 mM Hepes, pH8.0, 150
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mM NaCl, 0.25% NP-40, 10% glycerol and 5 mM MgCl2). The lysates were precleared by
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centrifugation and then incubated with streptavidin beads (Sigma) for 3 h at 4°C, in the
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presence of RNase inhibitor (200 U/mL). The beads were subsequently washed 8 times
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with lysis buffer, and bound components were eluted with biotin of 1 µg/mL. The eluted
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products were analyzed by urea PAGE for RNAs and SDS-PAGE for proteins,
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respectively. For SDS-PAGE, the gel was visualized by silver staining (Silver stain plus kit,
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Bio-Rad), and unique protein bands were cut off for mass spectrometry analyses
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(LC-MS/MS).
co-transfection
of
DROSHA
siRNA-encoding
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plasmid
together
with
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Biochemistry
Immunoprecipitation and Western blot
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Cells were washed with PBS, and then extracted with lysis buffer (20 mM Tris-HCl pH
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7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Tween 20, and Roche’s complete protease
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inhibitors) and centrifuged at 15,000 g for 10 min at 4°C. The proteins in the supernatant
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were measured using Bradford reagent (Sigma)
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500 µg of total cell lysates were incubated with anti-Myc or anti-Flag beads (Sigma) for 2
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h at 4°C. The bound proteins were eluted with 400 µg/mL synthetic Myc or 3×Flag
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peptides, respectively.
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21
. For immunoprecipitation analysis,
For Western blot, experimental samples were separated by electrophoresis on 10%-15%
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SDS-PAGE, and transferred onto a PVDF membrane (Millipore Corp.). After blocking
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with PBST containing 5% skim milk, membranes were incubated with primary antibodies,
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followed by incubation with horseradish peroxidase-conjugated secondary antibodies,
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and developed using Millipore’s chemiluminescence substrate. To determine the
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equivalence of protein amounts loaded among different samples, the developed
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membranes were stripped with a buffer consisting of 62.5 mM Tris-HCl (pH 6.7), 2% SDS
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and 100 mM 2-mercaptoethanol for 1 h, followed by incubation with control antibodies for
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further blotting. In some cases, immunoblots were quantified by measuring the
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immunoreactive protein band density with software ImageJ 1.48 (NIH, USA)
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antibodies used in Western blot includes: anti-c-Myc (1:5000, C3956, Sigma); anti-Flag
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(1:5000, F1804, Sigma); anti-La (1:1000, sc-166274, Santa Cruz Biotechnology);
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anti-DROSHA (1:1000, 3364, Cell Signaling Technologies); anti-Dicer (1:1000, 5362, Cell 7
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. Primary
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Signaling Technologies) and anti-β-actin (1:5000, AV40173, Sigma).
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Real-time PCR
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Total RNA was extracted using Trizol reagent following the manufacturer’s instructions
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(Invitrogen), and total RNA (2 µg) was reversely transcribed to cDNAs using SuperScript
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II reverse transcriptase (Invitrogen). Quantification of mRNA levels was measured using
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real-time PCR (ABI Prism7500, Applied Biosystems) and SYBR Green qPCR Master Mix
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(KAPA Biosystems). The primers are shown in supplemental data (Table S1). Specific
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quantification of miRNA levels was performed using Universal ProbeLibrary probe-21
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assay (Roche) as described previously 23. Briefly, the reaction conditions consisted of 1 µl
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of cDNA and 0.4 µM primers in a final volume of 20 µl of supermix. For mRNA, each cycle
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consisted of denaturation at 95°C for 15s, annealing at 55°C for 30s and extension at
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72°C for 30s, respectively. For miRNA, each cycle consisted of denaturation at 95°C for
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15s, annealing at 58°C for 30s. The sequences of all miRNA precursors were used to
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design specific forward and stem-loop RT primers which can be found in the
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supplementary data (Table S1). microRNA was normalized to snoRNA-U6 under the
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same conditions.
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Transfection and gene silencing by shRNA
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5×105 cells were seeded on 60-mm tissue culture dishes and cultured overnight. 8 µg
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of La/SSB shRNA plasmids, which was purchased from Axil Scientific Pte Ltd 8
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(Singapore), were transfected into cells using PEI as transfection reagent individually.
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siRNA for DROSHA/Dicer and scramble control siRNA were also purchased from Axil
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Scientific Pte Ltd (Singapore). Expression level of the target protein modulated by gene
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silencing or overexpression was determined by Western blot using corresponding
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primary antibodies 2 days after transfection.
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Electrophoretic mobility shift assay
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Pri-miRNAs containing digoxygenin-labeled rUTP (Roche) were transcribed in vitro,
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using RiboMAX Large Scale RNA Production Kit (Promega). The transcribed
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oligoribonucleotides were incubated at room temperature with 1 microgram different
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proteins in the buffer containing 10 mMTris, pH 7.5, 50 mMKCl, 1 mM DTT, 10 mM MgCl2,
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0.1% NP-40 and 5% glycerol to a total reaction volume of 20 µL. Following 10 min
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incubation, the samples were immediately loaded onto 4% native polyacrylamide gel with
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non-denaturating dye. The resolved nucleic acids were electro-blotted onto Hybond-N+
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(GE healthcare) and cross-linked by UV. Blocking, detection and washing of the
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membrane were performed according to the instruction of DIG Gel Shift Kit (Roche).
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Protein expression and purification
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DNA encoding La protein and its truncations were amplified by PCR using cDNAs of
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human HEK293 cells. The expression constructs were generated by insertion of the PCR
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products into pET-28b/28m vector (Novagen) in frame with a carboxy-terminal 9
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hexahistidine-tag. Mutant alleles were prepared using QuickChange Site-Directed
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Mutagenesis Kit (Stratagene) and verified by sequencing. Recombinant La protein,
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truncations and its mutants were expressed in E. coli (strain BL21/DE3) overnight at 20°C
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induced by 0.4 mM isopropyl β-D-thiogalactoside. The proteins were purified with Ni2+
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affinity column, followed by HiLoad Superdex S-75 26/60 column.
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In vitro stem-loop miRNA precursors processing assay and absolute quantitation
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The in vitro processing method has been previously described24. Microprocessor
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containing Flag-DROSHADGCR8DicerAgo2 was extracted and purified from HEK293
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stable cell lines and HEK293 Dicer- (dicer knocked-down by shRNA) cells using
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anti-FLAG M2 affinity gel (Sigma) , respectively. After washing with buffer A (20 mM
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Tris-HCl pH 7.9, 0.5 M KCl, 10% glycerol, 1 mM EDTA, 5 mM DTT, and 0.2 mM PMSF
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0.5% NP-40) twice, followed by washing with buffer B (20 mMTris-HCl pH 7.9, 0.1 M KCl,
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10% glycerol, 1 mM EDTA, 5 mM DTT, and 0.2 mM PMSF) once, the bound proteins
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were eluted from the affinity column by synthetic 3×FLAG peptide. In vitro processing
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reactions were performed in a final volume of 10 µL by mixture of 1 microgram in vitro
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transcribed stem-loop miRNA precursors and purified microprocessor containing
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Flag-DROSHA, FDGCR8 and Dicer or microprocesor containing Flag-DROSHA and
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DGCR8 for 90 min at 37°C in a buffer containing 3.2 mM MgCl2, 1 U/µL RNasin, 20 mM
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Tris-HCl pH 7.9, 0.1 M KCl, and 10% glycerol
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RNA was precipitated and resolved in DEPC water, which was then used as the template
25
. Reactions were phenol-extracted and
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for Taqman qRT-PCR as previously described. Meanwhile, three sets of equal copies
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synthesized siRNA 21 were prepared and 10-fold serial dilutions of siRNA 21were used
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for RT-PCR and Taqman specific qRT-PCR. The resulting Ct values of the synthetic
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miRNAs were used to generate a standard curve to calculate in vitro cleavage miRNA
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copy number. The copy number for each miRNA was individually calculated using
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different sets of standard curve (Fig. S1C-E).
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Northern blotting and MicroRNA microarray 26
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Northern blotting for miRNA detection was performed essentially as described
.
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Typically, 20 µg of total RNA was resolved by 12% urea-PAGE and transferred to Hybond
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N+ membrane followed by UV cross-linking. All miRNA probes are 21-nt or 20-nt ssDNAs
209
that are complementary to miRNA sequences provided by miRBase. The U6 probe
210
sequence is 5’-ACGAATTTGCGTGTATCCTT. All probes were 5′ biotin-labeled.
211
Hybridization was conducted in ultrasensitive hybridization buffer (Ambion) at 42°C
212
overnight. The membrane was washed 3 times in 2× SSC, 0.1% SDS, and exposed.
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HEK293 cells were transfected with La/SSB shRNA plasmids as described previously
214
and repeated three times. RNA was extracted and used for microRNA microarray.
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MicroRNA microarray service supplied by Ribobio was used for genome-wide miRNA
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expression profiling. Ribobio (www.ribobio.com) provides a genome-wide miRNA
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(miRNA) expression profiling service using a GenePix 4000B scanner and Agilent 2200
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Bioanalyzer. Microarray analyses were repeated three times each.
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Melting curve analysis of the pri-let-7 miRNA
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Normalized UV absorbance of the pri-let-7 in the absence and presence (pri-let-7 + La)
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of La protein, were monitored as samples were gradually heated from 25 ℃ to 90 ℃ at a
223
rate of 1 ℃ per minute. RNA and protein were at a concentration of 5 µM and in a buffer
224
containing 20 mM sodium cacodylate, pH6.5, 2.5 M MgCl2, and 2 mM spermidine. The
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UV melting experiments were run on a Jasco-J-810 spectrophotometer connected to a
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PC for data acquisition27.
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Results
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La protein is associated with pri-let-7a
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To identify the putative protein components directly involved in primary miRNA
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processing in human system, we modified the developed tRNA-scaffolded streptavidin
232
aptamer (tRSA) vector by fusion of primary let-7a miRNA sequence (402 nt in total) at the
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3’-end of the scaffold (Fig. 1A)
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sequences, driven by CMV promoter, was transfected into HEK293 cells and its RNA
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transcript was used to pull down proteins associated with pri-let-7a (Fig. 1B). To avoid the
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cleavage of pri-let-7a by DROSHA in cells, plasmid encoding DROSHA-specific siRNA
237
was co-transfected into HEK293 cells (Fig.S1A). To eliminate the possibility that
238
DROSHA siRNA may have off-target effect, which could potentially affect the DGCR8
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stability, we performed western blot and found the expression level of DGCR8 remained
17
. The plasmid encoding tRSA-tagged pri-let-7a
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Biochemistry
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unchanged (Fig.S1A). After 48 hours of culture, the transfected cells were harvested and
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the cell extracts were incubated with streptavidin beads. The tRSA vehicle and tRSA in
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fusion with pri-let-7a were successfully eluted with biotin (Fig. 1C), which demonstrated
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that tRSA and tRSA in fusion with pri-let-7a were efficiently expressed in HEK293 cells
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and easily purified by pull-down assay. The co-existence of tRSA and tRSA-pri-let-7a (Fig.
245
1C, third column) may suggest that the knockdown of DROSHA by siRNA does not
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completely eliminate DROSHA expression and/or RNA transcription might be partially
247
digested after tRSA-pri-let-7a was transcribed. Nevertheless, the distinct tRSA-pri-let-7a
248
band revealed by Northern blot demonstrated that pri-let-7a was successfully expressed
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and isolated in this assay.
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Figure 1. La protein is associated with pri-let-7a in cells. (A) Schematic of the tRNA-scaffolded
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streptavidin aptamer (tRSA) constructed for this assay. Bait RNAs are attached to the 3′-end of a
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transfer RNA-SA fusion. (B) Schematic of the construction of pri-miRNA expression plasmid
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pcDNA5/tRSA-pri-let-7a. (C-E) Identification of specific pri-let-7a miRNA-interacting proteins by
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tRSA affinity tag. Pull-downs were performed for HEK293 whole cell lysates, using tRSA (Veh) or
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tRSA-pri-let-7a (let-7a) as baits. Captured RNAs were analyzed by Northern blot, detected with
257
tRSA-specific probe (C). Captured proteins were analyzed by silver staining (D) and further
258
confirmed by Western blot with anti-La antibody (E).
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259 260
Meanwhile, we examined the RNA-binding proteins in SDS-PAGE visualized by silver
261
staining. Compared to tRSA control, one apparent abundant band of approximate 50 kDa
262
was observed in SDS-PAGE loaded with tRSA-pri-let-7a/protein sample (Fig. 1D). Next,
263
this highly accumulated protein band was identified as autoantigen La with 48 kDa by
264
mass spectrometry analysis, which is shown in supplementary data (Table S2).
265
To further validate the mass spectrometric result, anti-La antibody was used for
266
Western
blot
analysis.
As
expected,
a
distinct
band
was
detected
267
tRSA-pri-let-7a/protein sample and no obvious signal was detected in tRSA/protein
268
sample as the control (Fig. 1E). Our experiments strongly suggest that La protein could
269
specifically bind to pri-let-7a in cells. Meanwhile, these data demonstrate that tRSA fused
270
with a given RNA sequence could be used as a general strategy to detect and isolate
271
proteins particularly binding to the RNA sequence.
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La protein silencing affects stem-loop miRNA precursors processing in cells
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in
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Figure 2. La protein silencing affects stem-loop miRNA precursors processing in cells. (A) HEK293
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cells were transfected with shCTRL or shLa plasmids and the expression level of La protein was
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detected by Western blot. (B) Northern blot was performed to examine endogenous
278
miR-let-7a-5p and U6 snRNA was used as a loading control. (C) Heat map of miRNA microarray
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data in shLa-mediated La knock down HEK293 cells with fold changes >1.5 and an adjusted P
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value 1.5 and an adjusted P value
