MicroRNAs (miRNAs) are small noncoding RNAs which control gene expression by inhibiting mRNA translation. To date, >1000 miRNAs have been identified in humans and appear to target ~60% of human genes. Among them, let-7 miRNA is highly conserved from nematode to humans and involved in various biological processes. For example, let-7 functions as a tumor suppressor through oncogene repression. let-7 also regulates cell differentiation, developmental timing and apoptosis. Recently, it was discovered that precursor let-7 (pre-let-7) is polyuridylated at its 3' terminus by terminal uridyltransferase in several cancer cell lines and ES cells. Polyuridylated pre-let-7 is a poor substrate for Dicer and rapidly degraded by exonuclease Dis3l2. Thus, polyuridylation of pre-let-7 results in decreased expression of mature let-7 and contributes to tumorigenesis and ES cell maintenance.

Polyuridylation of pre-let-7 is performed by RNA binding protein Lin28 and terminal uridyltransferase-4 (TUT4) or TUT7. However, whether other factor(s) is involved or not is currently unknown. In this study, we set out to identify TUT4/7-associated proteins in search of novel factor(s) which regulate pre-let-7 polyuridylation. To this end, TUT4/7 complex was affinity-purified from doxycycline-inducible TUT4/7 stable cell line and analyzed by LC/MS/MS. Among the list of identified proteins is PARP13, one of human PARP homologues that are responsible for synthesis of poly(ADP) ribose (PAR) and attachment of PAR on acceptor proteins. The interaction between PARP13 and TUT4/7 prompted us to speculate that TUT4/7 could be modified and regulated by PAR.

In fact, we found that PAR was associated with TUT4/7 and diminished pre-let-7 polyuridylation in vitro. Furthermore, siRNA-mediated knockdown of the gene encoding PAR degrading enzyme leads to defective pre-let-7 polyuridylation in human cells. Moreover, when we treated cells with oxidative stress which is known to activate PARP, the amount of PAR associated with TUT4/7 was increased, while pre-let-7 polyuridylaiton was severely inhibited.

Taken together, these results suggest that PAR formed in response to oxidative stress binds to TUT4/7 to negatively regulate pre-let-7 polyuridylation. This is a new example of expanding roles of PAR in post-transcriptional gene regulation and provides insight into cellular stress response.