Tudor-SN–mediated endonucleolytic decay of human cell microRNAs promotes G ₁ /S phase transition

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Compared to miRNA biogenesis, pathways that mediate mature miRNA decay are less-well understood. We report that biologically functional miRNAs are degraded in human cells by the endonuclease Tudor-SN (TSN). In vitro, recombinant TSN initiates the decay of both protein-free and AGO2-loaded miRNAs via endonucleolytic cleavage at CA and UA dinucleotides, preferentially at scissile bonds located > five nucleotides from miRNA ends. Consistent with this, cellular targets of TSN-mediated decay defined using miR-seq follow this rule. Inhibiting TSN-mediated miRNA decay by CRISPR-Cas9 knockout of TSN inhibits cell-cycle progression by upregulating a cohort of miRNAs that downregulates mRNAs encoding proteins such as CDK2, CCND1, E2F1 and E2F2, which are critical for G1-to-S phase transition.

Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay

“…Our findings agree with the sequence alignment results, which suggest that SN1 and SN3 likely bear endonuclease activities (Li et al, 2008). However, they are not consistent with a truncation study showing that SN1 and SN4 in hTSN are crucial for endonuclease activity (Elbarbary et al, 2017b) (see Discussion).…”

“…It has been shown that TSN degrades certain hyper-edited primary miRNA precursors (Yang et al, 2006), as well as a group of mature miRNAs with a preference to cleave at UA and CA dinucleotides (Elbarbary et al, 2017b). Inosine editing may loosen the base pairing of miRNA precursors and increase their exposure to degradation by TSN.…”

“…Inosine editing may loosen the base pairing of miRNA precursors and increase their exposure to degradation by TSN. A recent study further shows that TSN interacts with UPF1 helicase which can dissociate miRNA from their mRNA targets, making the mature single-stranded miRNAs susceptible to the TSN-mediated miRNA decay (TumiD) (Elbarbary et al, 2017a (Elbarbary et al, 2017b). As SN1-SN2 and SN3-SN4 are assembled respectively into a structural lobe (see Figure 6), deletion of SN1 or SN4 might disrupt the folding of each lobe and impair TSN's ribonuclease activity, similar to our results of mutations in SN1 and SN3 domains.…”

“…-dependent RNase cleaving at the 5'-side of a phosphodiester bond As the endonuclease activity of hTSN is promoted by calcium ions (Elbarbary et al, 2017b), we first examined the metal ion-dependent activity of cTSN. We prepared a stem-loop RNA, named pre-miR142, which shares a similar structure and sequence to the pre-miR142 precursor that was used in a previous report (Yang et al, 2006).…”

“…Recently, TSN was further identified to be a ribonuclease that degrades both protein-free and Argonaute 2-loaded mature miRNA that promotes the G1/S phase transition in human cells (Elbarbary et al, 2017b). TSN therefore plays an important role in miRNA decay in mammals, targeting not only hyper-edited miRNA precursors, but also mature miRNA.…”