SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis

Haihua Yuan,Bin Jiang,Xian Zhao,Rong Deng,Guofang Hou,Hailong Zhang,Jianxiu Yu,Ming Xu,Ran Chen,Yanli Wang

doi:10.1186/s12943-017-0724-6

Abstract

BackgroundMicroRNAs (miRNAs) are important regulators involved in diverse physiological and pathological processes including cancer. SUMO (small ubiquitin-like modifier) is a reversible protein modifier. We recently found that SUMOylation of TARBP2 and DGCR8 is involved in the regulation of the miRNA pathway. KHSRP is a single stranded nucleic acid binding protein with roles in transcription and mRNA decay, and it is also a component of the Drosha-DGCR8 complex promoting the miRNA biogenesis.MethodsThe in vivo SUMOylation assay using the Ni2+-NTA affinity pulldown or immunoprecipitation (IP) and the in vitro E.coli-based SUMOylation assay were used to analyze SUMOylation of KHSRP. Nuclear/Cytosol fractionation assay and immunofluorescent staining were used to observe the localization of KHSRP. High-throughput miRNA sequencing, quantantive RT-PCR and RNA immunoprecipitation assay (RIP) were employed to determine the effects of KHSRP SUMO1 modification on the miRNA biogenesis. The soft-agar colony formation, migration, vasculogenic mimicry (VM) and three-dimensional (3D) cell culture assays were performed to detect the phenotypes of tumor cells in vitro, and the xenograft tumor model in mice was conducted to verify that SUMO1 modification of KHSRP regulated tumorigenesis in vivo.ResultsKHSRP is modified by SUMO1 at the major site K87, and this modification can be increased upon the microenvironmental hypoxia while reduced by the treatment with growth factors. SUMO1 modification of KHSRP inhibits its interaction with the pri-miRNA/Drosha-DGCR8 complex and probably increases its translocation from the nucleus to the cytoplasm. Consequently, SUMO1 modification of KHSRP impairs the processing step of pre-miRNAs from pri-miRNAs which especially harbor short G-rich stretches in their terminal loops (TL), resulting in the downregulation of a subset of TL-G-Rich miRNAs such as let-7 family and consequential tumorigenesis.ConclusionsOur data demonstrate how the miRNA biogenesis pathway is connected to tumorigenesis and cancer progression through the reversible SUMO1 modification of KHSRP.

Highlights

MicroRNAs are important regulators involved in diverse physiological and pathological processes including cancer
hnRNP K homology-type splicing regulatory protein (KHSRP) is SUMOylated in cells and in vitro To detect whether KHSRP can be SUMOylated in cells, we co-transfected His-SUMO1, Flag-Ubc9 and EBGSENP1 with (Fig. 1a) or without (Fig. 1b) HA-KHSRP plasmid into 293T cells, and performed the SUMOylation assay by using the method of Ni2+-NTA resin pulldown [28]
The SUMO1 modification of KHSRP was strongly enhanced by the E2 conjugating enzyme Ubc9, whereas it was attenuated with the addition of the deSUMOylation enzyme SENP1

Summary

Introduction

MicroRNAs (miRNAs) are important regulators involved in diverse physiological and pathological processes including cancer. KHSRP is a single stranded nucleic acid binding protein with roles in transcription and mRNA decay, and it is a component of the Drosha-DGCR8 complex promoting the miRNA biogenesis. High-throughput miRNA sequencing, quantantive RT-PCR and RNA immunoprecipitation assay (RIP) were employed to determine the effects of KHSRP SUMO1 modification on the miRNA biogenesis. The soft-agar colony formation, migration, vasculogenic mimicry (VM) and three-dimensional (3D) cell culture assays were performed to detect the phenotypes of tumor cells in vitro, and the xenograft tumor model in mice was conducted to verify that SUMO1 modification of KHSRP regulated tumorigenesis in vivo. MiRNAs are firstly transcribed by RNA polymerases II and III as primary miRNAs (pri-miRNAs) [1] These pri-miRNAs are later processed to the 65-nucleotide (nt) hairpin precursor miRNAs (pre-miRNAs) by the microprocessor complex (MC), which is mainly composed of Drosha and DGCR8 [2]. The mature miRNA, one strand of the duplex, and Argonaute (Ago) proteins mainly constitute a RNAinduced silencing complex (RISC), which mediates posttranscriptional gene silencing [5, 6]

Results

Discussion

Conclusion