Determinants of MicroRNA Processing Inhibition by the Developmentally Regulated RNA-binding Protein Lin28

Abstract

The developmentally regulated RNA-binding protein Lin28 blocks processing of let-7 family microRNAs (miRNAs) in embryonic cells. The molecular basis for this selective miRNA processing block is unknown. Here we find that Lin28 selectively binds the terminal loop region of let-7 precursors in vitro and that the loop mediates miRNA processing inhibition in vivo. Additionally, we identify the domains of Lin28 required for this inhibition. These findings establish a regulatory role for the terminal loop of precursors in miRNA maturation and provide insight into the mechanism by which Lin28 negatively regulates let-7 processing. MicroRNAs (miRNAs)2 comprise a large family of short regulatory RNAs that repress the expression of target messenger RNAs and have many important roles in development (1). In addition to the requirement of miRNAs for normal development, it is emerging that altered miRNA expression is a hallmark of various cancers (2). Several examples of miRNAs with oncogenic or tumor suppressor properties have been reported. Notably, let-7 miRNA has been reported to play a tumor suppressor role by repression of oncogenes including Hmga2, Ras,. Reduced expression of let-7 miRNA in human lung cancers is associated with shortened postoperative survival (7), and in a mouse lung cancer model, let-7g inhibits tumor development (8). Additionally, low let-7 expression is important for the self-renewal and tumorigenicity of breast cancer initiating cells (9).Hundreds of miRNAs have now been identified, many of which are expressed in a tissue-and developmental stage-specific manner. Under most conditions, control of their expression occurs at the transcriptional level. The miRNA biogenesis pathway involves the sequential processing of primary miRNA transcripts (pri-miRNAs) by the Microprocessor complex (comprising the RNaseIII enzyme Drosha and the doublestranded RNA-binding protein DGCR8) to release 60 -70-nt precursor miRNAs (pre-miRNAs) that are subsequently cleaved by the Dicer complex to yield mature ϳ22 nt miRNAs (10 -13). Emerging evidence indicates that miRNA biogenesis can also be regulated posttranscriptionally (14 -18).The developmentally regulated RNA-binding protein Lin28 was recently identified as a selective inhibitor of miRNA processing in embryonic stem cells and embryonal carcinoma cells (18). Lin28 inhibits the maturation of the let-7 family but not other miRNAs, yet a mechanistic explanation for this selectivity is unknown. We sought to gain insight into the mechanism by which Lin28 selectively blocks the processing of let-7 family miRNAs. Using in vitro and in vivo assays, we explored the RNA sequence and structural requirements for Lin28-mediated regulation and found that Lin28 specifically binds the terminal loop region of let-7 precursors. Furthermore, we demonstrated that the loop mediates miRNA processing inhibition in vivo and identified the domains of Lin28 required for this inhibition. EXPERIMENTAL PROCEDURESElectromobilty Shift Assays (EMSA)-EMSA was conducted using ϳ2 ϫ 10 5 cpm 5Ј-end-l...

A genetic variant in the pre-miR-27a oncogene is associated with a reduced familial breast cancer risk

“…Consequently the cleavage by Drosha would be blocked, and the maturation process is thereby inhibited. Piskounova et al showed that some Drosha inhibitors such as Lin28, selectively bind to the terminal loop region of miRNA precursors and mediate the miRNA procession [53,54]. Thus, it is also possible that the [A]-[G] variant changes the binding affinity of some Drosha inhibitors and consequently affects the processing of miR-27a.…”

Lin-28B transactivation is necessary for Myc-mediated let-7 repression and proliferation

“…An additional Lin-28 homolog, Lin-28B, was identified as a highly expressed gene in hepatocellular carcinoma cells (41). Recently, Lin-28 and Lin-28B were demonstrated to function as negative regulators of let-7 biogenesis (30)(31)(32)(33)(34). These proteins are members of a highly conserved family of orthologous RNA binding proteins that contain a cold shock domain and dual zinc fingers.…”

Role of Ribosomal Protein RPS2 in Controlling let-7a Expression in Human Prostate Cancer

“…Piskounova et al (50) have clearly shown that LIN28 selectively binds the terminal loop region of precursor let-7g miRNAs (i.e., both pri-and pre-let-7g sequences) and mature let-7g localized primarily to the cytoplasm (51). It has also been shown that the expression of LIN28B and less frequently LIN28 is activated in several cancer types and their overexpression causes cellular transformation (6) along with increased c-Myc, HMGA2, and ras expression (52).…”