Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition

Human LIN28A and LIN28B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ∼3000 mRNAs at ∼9500 sites located in the 3′ UTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors.     

let-7 microRNAs in development, stem cells and cancer

MicroRNAs (miRNAs) are small noncoding RNAs, approximately 22 nucleotides in length, that repress target messenger RNAs (mRNAs) through an antisense mechanism. The let-7 miRNA was originally discovered in the nematode Caenorhabditis elegans, where it regulates cell proliferation and differentiation, but subsequent work has shown that both its sequence and its function are highly conserved in mammals. Recent results have now linked decreased let-7 expression to increased tumorigenicity and poor patient prognosis. Moreover, during normal development, accumulation of let-7 can be prevented by LIN28, a promoter of pluripotency. Based on these findings, we propose that let-7 regulates 'stemness' by repressing self-renewal and promoting differentiation in both normal development and cancer. A more complete understanding of its function will thus provide insights into these processes and might yield diagnostic and therapeutic advances for cancer treatment.     

Ectopic over-expression of tristetraprolin in human cancer cells promotes biogenesis of let-7 by down-regulation of Lin28

Tristetraprolin (TTP) is a AU-rich element (ARE) binding protein and exhibits suppressive effects on cell growth through down-regulation of ARE-containing oncogenes. The let-7 microRNA has emerged as a significant factor in tumor suppression. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. In this work, an unexpected link between TTP and let-7 has been found in human cancer cells. TTP promotes an increase in expression of mature let-7, which leads to the inhibition of let-7 target gene CDC34 expression and suppresses cell growth. This event is associated with TTP-mediated inhibition of Lin28, which has emerged as a negative modulator of let-7. Lin28 mRNA contains ARE within its 3'-UTR and TTP enhances the decay of Lin28 mRNA through binding to its 3'-UTR. This suggests that the TTP-mediated down-regulation of Lin28 plays a key role in let-7 miRNA biogenesis in cancer cells.     

Lin28-mediated control of let-7 microRNA expression by alternative TUTases Zcchc11 (TUT4) and Zcchc6 (TUT7)

The pluripotency factor Lin28 recruits a 3' terminal uridylyl transferase (TUTase) to selectively block let-7 microRNA biogenesis in undifferentiated cells. Zcchc11 (TUTase4/TUT4) was previously identified as an enzyme responsible for Lin28-mediated pre-let-7 uridylation and control of let-7 expression. Here we investigate the protein and RNA determinants for this interaction. Biochemical dissection and reconstitution assays reveal the TUTase domains necessary and sufficient for Lin28-enhanced pre-let-7 uridylation. A single C2H2-type zinc finger domain of Zcchc11 was found to be responsible for the functional interaction with Lin28. We identify Zcchc6 (TUTase7) as an alternative TUTase that functions with Lin28 in vitro, and accordingly, we find Zcchc11 and Zcchc6 redundantly control let-7 biogenesis in embryonic stem cells. Our study indicates that Lin28 uses two different TUTases to control let-7 expression and has important implications for stem cell biology as well as cancer.     

Drosha mediates destabilization of Lin28 mRNA targets

Lin28 plays important roles in development, stem cell maintenance, oncogenesis and metabolism. As an RNA-binding protein, it blocks the biogenesis primarily of let-7 family miRNAs and also promotes translation of a cohort of mRNAs involved in cell growth, metabolism and pluripotency, likely through recognition of distinct sequence and structural motifs within mRNAs. Here, we show that one such motif, shared by multiple Lin28-responsive elements (LREs) present in Lin28 mRNA targets also participates in a Drosha-dependent regulation and may contribute to destabilization of its cognate mRNAs. We further show that the same mutations in the LREs known to abolish Lin28 binding and stimulation of translation also abrogate Drosha-dependent mRNA destabilization, and that this effect is independent of miRNAs, uncovering a previously unsuspected coupling between Drosha-dependent destabilization and Lin28-mediated regulation. Thus, Lin28-dependent stimulation of translation of target mRNAs may, in part, serve to compensate for their intrinsic instability, thereby ensuring optimal levels of expression of genes critical for cell viability, metabolism and pluripotency.     

Drosha mediates destabilization of Lin28 mRNA targets

Lin28 plays important roles in development, stem cell maintenance, oncogenesis and metabolism. As an RNA-binding protein, it blocks the biogenesis primarily of let-7 family miRNAs and also promotes translation of a cohort of mRNAs involved in cell growth, metabolism and pluripotency, likely through recognition of distinct sequence and structural motifs within mRNAs. Here, we show that one such motif, shared by multiple Lin28-responsive elements (LREs) present in Lin28 mRNA targets also participates in a Drosha-dependent regulation and may contribute to destabilization of its cognate mRNAs. We further show that the same mutations in the LREs known to abolish Lin28 binding and stimulation of translation also abrogate Drosha-dependent mRNA destabilization, and that this effect is independent of miRNAs, uncovering a previously unsuspected coupling between Drosha-dependent destabilization and Lin28-mediated regulation. Thus, Lin28-dependent stimulation of translation of target mRNAs may, in part, serve to compensate for their intrinsic instability, thereby ensuring optimal levels of expression of genes critical for cell viability, metabolism and pluripotency.     

Divergent LIN28-mRNA associations result in translational suppression upon the initiation of differentiation

LIN28 function is fundamental to the activity and behavior of human embryonic stem cells (hESCs) and induced pluripotent stem cells. Its main roles in these cell types are the regulation of translational efficiency and let-7 miRNA maturation. However, LIN28-associated mRNA cargo shifting and resultant regulation of translational efficiency upon the initiation of differentiation remain unknown. An RNA-immunoprecipitation and microarray analysis protocol, eRIP, that has high specificity and sensitivity was developed to test endogenous LIN28-associated mRNA cargo shifting. A combined eRIP and polysome analysis of early stage differentiation of hESCs with two distinct differentiation cues revealed close similarities between the dynamics of LIN28 association and translational modulation of genes involved in the Wnt signaling, cell cycle, RNA metabolism and proteasomal pathways. Our data demonstrate that change in translational efficiency is a major contributor to early stages of differentiation of hESCs, in which LIN28 plays a central role. This implies that eRIP analysis of LIN28-associated RNA cargoes may be used for rapid functional quality control of pluripotent stem cells under manufacture for therapeutic applications.     

A LIN28-dependent structural change in pre-let-7g directly inhibits dicer processing

Several recent studies have provided evidence that LIN28, a cytoplasmic RNA-binding protein, inhibits the biogenesis of members of the let-7 microRNA family at the Dicer step in both mammals and Caenorhabditis elegans. However, the precise mechanism of inhibition is still poorly understood. Here we report on an in vitro study, which combined RNase footprinting, gel shift binding assays, and processing assays, to investigate the molecular basis and function of the interaction between the native let-7g precursor (pre-let-7g) and LIN28. We have mapped the structure of pre-let-7g and identified some regions of the terminal loop of pre-let-7g that physically interact with LIN28. We have also identified a conformational change upon LIN28 binding that results in the unwinding of an otherwise double-stranded region at the Dicer processing site of pre-let-7g. Furthermore, we showed that a mutant pre-let-7g that displays an open upper stem inhibited pre-let-7g Dicer processing to the same extent as LIN28. The data support a mechanism by which LIN28 can directly inhibit let-7g biogenesis at the Dicer processing step.     

LIN28B regulates androgen receptor in human trophoblast cells through Let‐7c

LIN28B is an RNA‐binding protein necessary for maintaining pluripotency in stem cells and plays an important role in trophoblast cell differentiation. LIN28B action on target gene function often involves the Let‐7 miRNA family. Previous work in cancer cells revealed that LIN28 through Let‐7 miRNA regulates expression of androgen receptor (AR). Considering the similarities between cancer and trophoblast cells, we hypothesize that LIN28B also is necessary for the presence of AR in human trophoblast cells. The human first‐trimester trophoblast cell line, ACH‐3P was used to evaluate the regulation of AR by LIN28B, and a LIN28B knockdown cell line was constructed using lentiviral‐based vectors. LIN28B knockdown in ACH‐3P cells resulted in significantly decreased levels of AR and increased levels of Let‐7 miRNAs. Moreover, treatment of ACH‐3P cells with Let‐7c mimic, but not Let‐7e or Let‐7f, resulted in a significant reduction in LIN28B and AR. Finally, forskolin‐induced syncytialization and Let‐7c treatment both resulted in increased expression of syncytiotrophoblast marker ERVW‐1 and a significant decrease in AR in ACH‐3P. These data reveal that LIN28B regulates AR levels in trophoblast cells likely through its inhibitory actions on let‐7c, which may be necessary for trophoblast cell differentiation into the syncytiotrophoblast.     

Mammalian DIS3L2 exoribonuclease targets the uridylated precursors of let-7 miRNAs

The mechanisms of gene expression regulation by miRNAs have been extensively studied. However, the regulation of miRNA function and decay has long remained enigmatic. Only recently, 3′ uridylation via LIN28A-TUT4/7 has been recognized as an essential component controlling the biogenesis of let-7 miRNAs in stem cells. Although uridylation has been generally implicated in miRNA degradation, the nuclease responsible has remained unknown. Here, we identify the Perlman syndrome-associated protein DIS3L2 as an oligo(U)-binding and processing exoribonuclease that specifically targets uridylated pre-let-7 in vivo. This study establishes DIS3L2 as the missing component of the LIN28-TUT4/7-DIS3L2 pathway required for the repression of let-7 in pluripotent cells.     

Cellular microRNA let-7c inhibits M1 protein expression of the H1N1 influenza A virus in infected human lung epithelial cells

The influenza virus (IV) triggers a series of signalling events inside host cells and induces complex cellular responses. Studies have suggested that host factors play an essential role in IV replication. MicroRNAs (miRNAs) represent a class of small non-coding RNAs that target mRNAs, triggering either translation repression or RNA degradation. Emerging research suggests that host-derived cellular miRNAs are involved in mediating the host-IV interaction. Using miRNA microarrays, we identified several miRNAs aberrantly expressed in IV-infected human lung epithelial cells (A549). Specifically, miR-let-7c was highly up-regulated in IV-infected A549 cells. PITA and miRanda database screening indicated that the let-7c seed sequence is a perfect complementary sequence match to the 3' untranslated region (UTR) of viral gene M1 (+) cRNA, but not to PB2 and PA. As detected by a luciferase reporter system, let-7c directly targeted the 3'-UTR of M1 (+) cRNA, but not PB2 and PA. To experimentally identify the function of cellular let-7c, precursor let-7c was transfected into A549 cells. Let-7c down-regulated IV M1 expression at both the (+) cRNA and protein levels. Furthermore, transfection with a let-7c inhibitor enhanced the expression of M1. Therefore, let-7c may reduce IV replication by degrading M1 (+) cRNA. This is the first report indicating that cellular miRNA regulates IV replication through the degradation of viral gene (+) cRNA by matching the 3'-UTR of the viral cRNA. These findings suggest that let-7c plays a role in protecting host cells from the virus in addition to its known cellular functions.