February 2024
Authors:
Kyung-Won Min, Myung Hyun Jo, Minseok Song, Ji Won Lee, Min Ji Shim, Kyungmin Kim, Hyun Bong Park,Shinwon Ha, Hyejin Mun, Ahsan Polash, Markus Hafner, Jung-Hyun Cho, Dongsan Kim, Ji-Hoon Jeong, Seungbeom Ko, Sungchul Hohng, Sung-Ung Kang & Je-Hyun Yoon
Abstract:
“Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.“
Sage Science Products:
PippinHT was used to isolate micro RNA libraries.
Methods Excerpt:
“Library preparation was performed using the TruSeq Small RNA library preparation kit (Illumina, RS-200) following the manufacturer’s instructions. Briefly, 1 μg of input RNA was loaded into Urea-TBE gels for purification and the resulting RNAs were applied for the following procedures. User-supplied reagents including T4 RNA ligase2 Deletion Mutant (Lucigen, LR2D1132K) and Maxima First Stand cDNA synthesis kit (Thermo Fisher Scientific, K1641) were purchased separately. Libraries were amplified using 11 cycles of PCR for the manufacture’s index or modified index primer set to increase diversity. Libraries were prepared according to the manufacturer’s protocol with a modification in the size selection step, which, instead of agarose gel purification, PippinHT Prep instrument (Sage Science, HTP0001) and 3% agarose dye-free cassette with internal standards (Sage Science, HTG3010) was used under the following conditions: base pair start = 120 bp, base pair end = 160 bp, range = broad, target peak size = 145 bp. Eluted Libraries from PippinHT system were subsequently analysed on Tape-station 4150 (Agilent Technologies, G2992AA) following the manufacturer’s instructions using a High Sensitivity DNA Screen tape (Agilent Technologies, 5067–5584). Each library was barcoded with unique sequence of reverse primers during the PCR step, which contained Illumina compatible indices and modified indexes (see GEO database). Before pooling libraries for the next-generation sequencing, concentration of each library was measured in a high sensitivity Tape-station followed by smear analysis. Illumina NextSeq 550 or Miniseq with single end (50 nt;R1) read method was apply for the library sequencing.”
RNA Biology
DOI: 10.1080/15476286.2024.2314846