The ubiquitin ligase human TRIM71 regulates let-7 microRNA biogenesis via modulation of Lin28B protein

https://doi.org/10.1016/j.bbagrm.2014.02.017Get rights and content

Highlights

  • TRIM71 modulates ubiquitination and degradation of Lin28B.

  • C-terminus of Lin28B is essential for ubiquitination and interactions with TRIM71.

  • RING finger motif of TRIM71 is essential for interactions and let-7 biogenesis.

  • TRIM71 modulates let-7 miRNA that targets HMGA2 expression.

  • let-7 is part of a negative feedback loop that targets the TRIM71–3′UTR.

Abstract

let-7 microRNA (miRNA) is implicated in various biological processes, and its downregulation essentially linked to human malignancy. Regulation of gene expression of the let-7 family is critically linked to RNA-binding proteins. For instance, Lin28B and its paralog, Lin28A, inhibit the pre-let-7 precursor from being processed to mature miRNA by recruiting terminal uridyltransferase, TUT4, which adds oligomeric U at the 3′ end, suggesting that deregulation of Lin28B, together with Lin28A, may alter various biological processes through modulation of let-7 expression. Here, we showed that the Lin28B protein level is regulated via ubiquitin-mediated proteasomal degradation, and identified the ubiquitin ligase as human TRIM-NHL domain-containing TRIM71. In cells, TRIM71 negatively regulates Lin28B protein stability by catalyzing polyubiquitination. Compared with its paralog, Lin28A, a C-terminal unique ~ 50 amino acid stretch of Lin28B is essential for TRIM71 interactions and subsequent polyubiquitination. Moreover, the N-terminal RING finger motif of TRIM71 is critical for protein–protein interactions and polyubiquitination of Lin28B, and consequent let-7 expression. Consistent with the let-7 stimulatory role of TRIM71 via Lin28B polyubiquitination, specific knockdown of TRIM71 led to downregulation of let-7 expression. Expression of one of the known let-7 targets, HMGA2, was derepressed after knockdown of TRIM71. We additionally showed that enhanced expression of let-7 is part of a feedback loop that targets TRIM71 3′UTR, which contains two conserved let-7 target sites. Our findings collectively reveal critical aspects of regulatory complexity of let-7 biogenesis at the posttranscriptional level.

Introduction

MicroRNAs (miRNAs) are well known for their widespread function in development. Recent reports have implicated miRNAs in various diseases, most notably cancer [1]. Moreover, deregulation of miRNA expression appears to be directly linked with tumor formation, progression, and metastasis [2], [3]. The let-7 miRNA family was initially identified as a key regulator of gene expression during early development of Caenorhabditis elegans (C. elegans) [4]. These miRNAs are additionally renowned for their tumor suppressor activities through inhibition of key regulators, including high mobility group AT-hook 2 (HMGA2), RAS family, and c-Myc at the posttranscriptional level [4], [5], [6], [7], [8]. Expression levels of the let-7 family are generally downregulated in numerous cancers, and low levels are correlated with poor prognosis [4], [8].

Biogenesis of the let-7 family is mainly modulated by RNA-binding Lin28 proteins at the posttranscriptional level [9], [10], [11]. In contrast to C. elegans whereby a single Lin28 gene is responsible for regulation of let-7 expression, the vertebrate genome encodes two Lin28 paralogs [12], [13], [14]. Both Lin28A and Lin28B specifically interact with the loop sequence of pre-let-7 [9], [10], [15] and mediate terminal oligouridylation by recruiting the terminal uridyltransferase, TUT4 [9], [16], [17], [18]. Oligouridylated pre-let-7 is resistant to Dicer processing and susceptible to degradation [9]. However, these two paralogs were suggested to have different roles with regard to the posttranscriptional regulation of let-7 biogenesis [19]. Lin28A, which is mainly localized in the cytoplasm, faithfully mediates the oligouridylation and degradation of pre-let-7, whereas Lin28B, which is partially localized in the nucleus and predominantly localized in the cytoplasm [12], [20], sequesters pri-let-7 and blocks its further processing to the precursor form by the Drosha/DGCR8 microprocessor [19].

Mammalian Lin28B displays similar protein architecture to its paralog, Lin28A. However, Lin28B contains a unique ~ 50 amino acid serine/lysine-rich stretch in the carboxyl terminus (C-terminus) that appears prone to posttranslational modifications (PTMs). Tissue distribution and expression of Lin28B are abundant in placenta, testis, and fetal liver [12]. Notably, Lin28B is frequently overexpressed in hepatocellular carcinoma (HCC) [12], colorectal cancer [21], [22], and related with induction of neuroblastoma [23]. Moreover, ectopic expression of Lin28B in NIH/3T3 cells stimulates cell transformation, possibly through repression of let-7 expression, supporting a potential oncogenic property of this protein [24].

Lin28B is believed to have other non-let-7 miRNA functions. Recent reports have also shown that Lin28B has let-7-independent functions in migration, invasion, and transformation by associating with and repressing a subset of mRNAs (e.g., LGR5 and PROM1) that are essential for colon cancer progression [22]. Moreover, Most recently, a photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis identified thousands of human mRNAs that show direct binding to Lin28B [20], [25]. Intriguingly, these reports revealed that Lin28B enhances global protein synthesis and stabilizes target mRNAs. Thus, although Lin28B is generally considered to be a key regulator of let-7 biogenesis, additional work will be required to define its precise roles and the posttranscriptional regulatory mechanisms underlying the fine-tuning of its activity.

In the current study, we have shown that human Lin28B activity is regulated at the posttranslational level via ubiquitin-mediated proteasomal degradation. To our knowledge, this is the first study to report posttranslational control of let-7-regulating human Lin28B. Furthermore, TRIM-NHL-containing TRIM71 has been identified as the specific ubiquitin ligase for Lin28B, and its effects on Lin28B activity, consequent let-7 biogenesis were determined.

Section snippets

Cell culture

Human embryonic kidney cell line 293T was obtained from the American Type Culture Collection. Human embryonal carcinoma Tera-1 cell line was obtained from the Korean Cell Line Bank (Seoul, Republic of Korea) or Hyonchol Jang (National Cancer Center, Republic of Korea). Human embryonal carcinoma NCCIT was obtained from Kyung-Soon Park (CHA University, Republic of Korea). Huh7 was obtained from Sung Key Jang (POSTECH, Republic of Korea). Mouse embryonal carcinoma P19 cell line was obtained from

Polyubiquitination of Lin28B in cells

It is widely accepted that the biological role of Lin28B is similar to that of its paralog, Lin28A, in terms of let-7 miRNA turnover [9], [16], [19]. Both Lin28A and Lin28B specifically interact with the loop sequence of pre-let-7 [9], [10], [15] and mediate terminal oligouridylation by recruiting the terminal uridyltransferase, TUT4. While the detailed embryonic role of Lin28A has been documented, the precise function of Lin28B remains to be clarified. Lin28A and Lin28B share structural

Discussion

In the current study, we investigated the role of human TRIM71 as a specific ubiquitin ligase for the let-7 modulator, Lin28B. Several previous reports suggested that TRIM71 may act as an ubiquitin ligase [32], [40], [41]. For example, Ago2 was shown to be specifically ubiquitinated by mTRIM71, and SHCBP1 has been suggested as a substrate of mTRIM71 [32], [40]. Notably, Ago2 is a key component of the RISC complex and was found to be associated with TRIM71 [32], [40], [42], [43]. This prompted

Acknowledgements

We specially thank for V. Narry Kim (Seoul National University, Republic of Korea) for various expression constructs for the miRNA processing machinery. We are also grateful to Soo-Youl Kim (National Cancer Center, Republic of Korea; COLO 205, M14, NCI-H1299, and NCI/ADR-RES cell lines), Yun-Hee Kim (National Cancer Center, Republic of Korea; HepG2 cell line), Seok Hyun Kim (National Cancer Center, Republic of Korea; HA-Ub construct), Kyungsil Yoon (National Cancer Center, Republic of Korea;

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