Autoantigen La Regulates MicroRNA Processing from Stem–Loop

Oct 31, 2017 - Here, we utilize a newly developed RNA affinity technique to isolate such factors. In this study, a tRNA-scaffolded aptamer (tRSA)-base...
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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]

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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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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

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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

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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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. 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-DROSHA—DGCR8—Dicer—Ago2 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

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that are complementary to miRNA sequences provided by miRBase. The U6 probe

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sequence is 5’-ACGAATTTGCGTGTATCCTT. All probes were 5′ biotin-labeled.

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Hybridization was conducted in ultrasensitive hybridization buffer (Ambion) at 42°C

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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

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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

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rate of 1 ℃ per minute. RNA and protein were at a concentration of 5 µM and in a buffer

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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

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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

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was co-transfected into HEK293 cells (Fig.S1A). To eliminate the possibility that

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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.

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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

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digested after tRSA-pri-let-7a was transcribed. Nevertheless, the distinct tRSA-pri-let-7a

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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

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tRSA-specific probe (C). Captured proteins were analyzed by silver staining (D) and further

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confirmed by Western blot with anti-La antibody (E).

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Meanwhile, we examined the RNA-binding proteins in SDS-PAGE visualized by silver

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staining. Compared to tRSA control, one apparent abundant band of approximate 50 kDa

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was observed in SDS-PAGE loaded with tRSA-pri-let-7a/protein sample (Fig. 1D). Next,

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this highly accumulated protein band was identified as autoantigen La with 48 kDa by

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mass spectrometry analysis, which is shown in supplementary data (Table S2).

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To further validate the mass spectrometric result, anti-La antibody was used for

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Western

blot

analysis.

As

expected,

a

distinct

band

was

detected

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tRSA-pri-let-7a/protein sample and no obvious signal was detected in tRSA/protein

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sample as the control (Fig. 1E). Our experiments strongly suggest that La protein could

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specifically bind to pri-let-7a in cells. Meanwhile, these data demonstrate that tRSA fused

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with a given RNA sequence could be used as a general strategy to detect and isolate

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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

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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