SUMMARY When it escapes early detection, malignant melanoma becomes a highly-lethal and treatment-refractory cancer. Melastatin is greatly downregulated in metastatic melanomas and is widely believed to function as a melanoma tumor suppressor. Here we report that tumor suppressive activity is not mediated by melastatin but instead by a microRNA (miR-211) hosted within an intron of melastatin. Increasing expression of miR-211 but not melastatin reduced migration and invasion of malignant and highly invasive human melanomas characterized by low levels of melastatin and miR-211. An unbiased network analysis of melanoma-expressed genes filtered for their roles in metastasis identified three central node genes: IGF2R, TGFBR2, and NFAT5. Expression of these genes was reduced by miR-211 and knockdown of each gene phenocopied the effects of increased miR-211 on melanoma invasiveness. These data implicate miR-211 as a suppressor of melanoma invasion whose expression is silenced (or selected against) via suppression of the entire melastatin locus during human melanoma progression.

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 b s t r a c tUpon fusion of multivesicular bodies (MVBs) with the plasma membrane, intraluminal vesicles (ILVs) are released into the extracellular space as exosomes. Since the lipid composition of the exosomal membrane resembles that of raft microdomains, the inward budding process involves the raft-like region of the MVB limiting membrane. Although published research suggests that cellular RNAs may be selectively sorted into exosomes, the molecular mechanisms remain elusive. In this review, we suggest that there is a continuous interaction of cellular RNAs with the outer (cytoplasmic) surface of MVBs and that the selection for incorporation of these RNAs into ILVs is based on their affinity to the raft-like region in the outer layer of the MVB membrane.