Summary paragraph The pluripotency factor Lin28 inhibits the biogenesis of the let-7 family of mammalian microRNAs1–4. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development5, glucose metabolism6 and tissue regeneration7. Alternatively, Lin28 overexpression is correlated with the onset of numerous cancers8, while let-7, a tumor suppressor, silences several human oncogenes5. Lin28 binds to precursor let-7 (pre-let-7) hairpins9, triggering the 3' oligo-uridylation activity of TUT4/710–12. The oligoU tail added to pre-let-7 serves as a decay signal, as it is rapidly degraded by Dis3L213,14, a homolog of the catalytic subunit of the RNA exosome. The molecular basis of Lin28 mediated recruitment of TUT4/7 to pre-let-7 and its subsequent degradation by Dis3L2 is largely unknown. To examine the mechanism of Dis3L2 substrate recognition we determined the structure of mouse Dis3L2 in complex with an oligoU RNA to mimic the uridylated tail of pre-let-7. Three RNA binding domains form an open funnel on one face of the catalytic domain that allows RNA to navigate a path to the active site different from its exosome counterpart. The resulting path reveals an extensive network of uracil-specific interactions spanning the first twelve nucleotides of an oligoU-tailed RNA. We identify three U-specificity zones that explain how Dis3L2 recognizes, binds and processes uridylated pre-let-7 in the final step of the Lin28/let-7 pathway.

The uridyl transferases TUT4 and TUT7 switch between two modes of activity that either promotes let-7 expression (monoU) or marks it for degradation (oligoU). Lin28 modulates the switch via recruitment of TUT4 (7) to pre-let-7 in stem cells and human cancers. We found TUT4(7) utilize two multi-domain functional modules during the switch from mono-to oligoU. The catalytic module (CM) is essential for both activities, while the Lin28-interacting module (LIM) is indispensible for oligoU. The TUT7 CM structure trapped in the monoU state, revealed a duplex RNA binding pocket that orients group II pre-let-7 hairpins to position the 1-nt overhang favor monoU addition. Conversely, the switch to oligoU requires the ZK domain of Lin28 to drive the formation of a stable ternary complex between pre-let-7 and the inactive LIM. Finally, ZK2 of TUT4(7) aids oligoU addition by engaging the growing oligoU tail through uracil-specific interactions.The let-7 miRNA is broadly expressed in somatic cells and regulates cellular proliferation and differentiation, as well as the repression of several oncogenes and key regulators of mitogenic pathways, including HMGA2, MYC and RAS 1 . Humans have 12 let-7 variants (let-7a-1, -2, -3; let-7b-e; let-7f-1, -2; let-7g; let-7i; miR-98), with many tumors associated with the coordinated downregulation of multiple let-7 family members 2 . Mature let-7 is produced via the canonical miRNA biogenesis pathway, however its production levels are tightly controlled post-transcriptionally through the Lin28/let-7 pathway 3 . Elevated levels of Lin28 in stem cells 4 and a subset of human cancers 5-7 triggers the destruction of let-7 precursors [8][9][10][11][12] (pre-let-7) via recruitment of redundant terminal uridyltransferases, TUT4Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms Correspondence and requests for materials or additional data should be addressed to L.J (leemor@cshl.edu). 5 Current address: Plexxikon Inc., Berkeley, CA 94710 6 These authors contributed equally to this work Author contributions C.R.F., J.W. and L.J. designed and C.R.F and J.W. conducted all experiments. All authors contributed to data analysis and wrote the paper. Competing Financial Interests StatementThe authors declare no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript (ZCCHC11) and TUT7 (ZCCHC6), referred to collectively as TUT4(7). The oligoU tail added by TUT4(7) 13,14 is a signal for degradation of pre-let-7 by Dis3L2 [15][16][17] , which guarantees suppression of let-7 expression.As cells differentiate, Lin28 levels decline, causing TUT4(7) to switch their catalytic mode from processive oligouridylation to distributive monouridylation of group II pre-let-7 18 . Group II pre-let-7s (and other miRNAs) acquire a 1-nt overhang from Drosha processing ...

For the analysis of microRNA, a common approach is to first extract microRNA from cellular samples prior to any specific microRNA detection. Thus, it is important to determine the quality and yield of extracted microRNA. In this study, solid-phase extraction was used to isolate small RNA (<200 nt), which included microRNA, from mouse brain tissues. By using standard UV absorbance measurements, the amount of small RNA in the extracted RNA samples was determined. To determine the presence of microRNA, each RNA sample was analyzed by PAGE with SYBR^® Green II staining. Testing for contamination of any small DNA fragments, RNase and cellular peptides or proteins were systematically carried out. By scanning the gel image obtained from PAGE analysis, the average percentage of total microRNA (19 - 25 nt) in the extracted RNA samples was determined to be equal to 2.3 ± 0.5%. The yield of total microRNA was calculated to be ~0.5ng of microRNA per milligram of frozen mouse brain tissue. In comparison to other methods that require the use of expensive specialized instrumentation, the approach of combining the standard UV absorbance and PAGE analysis represents a simple and viable method for evaluating the quality and yield of microRNA extraction from tissue samples

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