Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
The ubiquitin ligase human TRIM71 regulates let-7 microRNA biogenesis via modulation of Lin28B protein
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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2021, Biochimica et Biophysica Acta - Gene Regulatory MechanismsThe stability and oncogenic function of LIN28A are regulated by USP28
2019, Biochimica et Biophysica Acta - Molecular Basis of DiseaseCitation Excerpt :LIN28 undergoes post-translational modifications that affect stability and its protein level. A study demonstrated that ubiquitin ligase human TRIM71 polyubiquitinates and degrades LIN28B protein [21]; however, to the best of our knowledge, no study has reported on the ubiquitination and stability of LIN28A protein. Protein ubiquitination events are widely reported to be reversed by counteracting enzymes known as deubiquitinating enzymes (DUBs).
The let-7/Lin28 axis regulates activation of hepatic stellate cells in alcoholic liver injury
2017, Journal of Biological ChemistryCitation Excerpt :These data further support our notion that increased Lin28B contributes to HSC activation and alcoholic liver fibrosis. let-7 miRNA can activate TRIM71, which targets Lin28 for ubiquitin-mediated proteasomal degradation, thus inhibiting Lin28 expression at both posttranscriptional and posttranslational levels (28). The Lin28B-let-7-positive feedback loop is discovered in human cancer development and progression and established that Lin28 (which is targeted by let-7) inhibits the biogenesis of let-7 family miRNAs to support Lin28 expression (29, 30).
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These authors contributed equally to this work.