Supplementary MaterialsSupplementary Information 41467_2018_6526_MOESM1_ESM. siRNAs for four tested human and mouse cell lines. Toxicity of these siRNAs stems from targeting survival genes with C-rich 3UTRs. The grasp tumor suppressor miRNA miR-34a-5p is usually harmful through such a G-rich 6mer seed and is upregulated in cells subjected to genotoxic stress. An analysis of all mature miRNAs suggests that during development most miRNAs developed to avoid guanine at the 5 end of the 6mer seed sequence of the guideline strand. In contrast, for certain tumor-suppressive miRNAs the guideline strand contains a G-rich harmful 6mer seed, presumably to eliminate malignancy cells. Introduction RNA interference (RNAi) is a form of post-transcriptional regulation exerted by 19C21 nt long double-stranded RNAs that negatively regulate gene expression at the mRNA level. RNAi-active guideline RNAs can come from endogenous siRNAs and micro(mi)RNAs. For an miRNA, the RNAi pathway begins in the nucleus with transcription of a main miRNA precursor (pri-miRNA)1. Pri-miRNAs ARHGEF11 are first processed by the Drosha/DGCR8 microprocessor complex into pre-miRNAs2, which are then exported from your nucleus to the cytoplasm by Exportin-53. Once in the cytoplasm, Dicer processes them further4,5 and these mature dsRNA duplexes are then loaded into Argonaute (Ago) proteins to form the RNA-induced silencing complex (RISC)6. The sense/passenger strand is usually ejected/degraded, while the guideline strand remains associated with the RISC7. Depending on the degree of complementarity between the guideline strand and its target, the outcome of RNAi can either be target degradationmost often achieved by siRNAs with full complementarity to their target mRNA8or miRNA-like cleavage-independent silencing, mediated by deadenylation/degradation or translational repression9. The latter mechanism can be initiated with as little as six nucleotide base-pairing between a guide RNAs so-called seed sequence (positions 2C7) and fully complementary seed matches in the target RNA10,11. This seed-based targeting most often occurs in the 3UTR of a target mRNA12,13. A number of miRNAs function either as tumor suppressors or as oncogenes14. Their cancer-specific activities are usually explained by their recognized targets, being oncogenes or tumor suppressors, respectively14. Examples of targets of tumor-suppressive miRNAs are the oncogenes Bcl-2 for miR-15/1615 and c-Myc for miR-34a16. While many miRNAs have been reported to have both tumor suppressive and oncogenic activities depending on the malignancy context, examples for widely established tumor-promoting miRNAs are miR-221/222, miR-21, miR-155, and users of the miR-17~92 cluster, or its paralogues miR-106b~25 and miR-106a~36317,18. In contrast, two of the major tumor-suppressive miRNA families are miR-15/16 and the p53 regulated miR-34a/c and miR-34b19. We recently discovered that many si- and shRNAs can kill all tested malignancy cell lines through RNAi by targeting the 3UTRs of crucial survival genes (SGs)20. We called this mechanism DISE (for death induced by ZJ 43 SG removal). Malignancy cells have difficulty in developing resistance to this mechanism both in vitro and when treated in vivo21. We reported that a 6mer seed sequence in the harmful siRNAs is sufficient for effective killing20. We have now performed a strand-specific siRNA screen with a library of individual siRNAs representing all 4096 possible ZJ 43 6mer seed sequences in a neutral RNA duplex. This screen, while based on siRNA biochemistry, was not designed to identify targets that are ZJ 43 degraded through siRNA-mediated slicing activity but to identify toxicity caused by moderately targeting hundreds ZJ 43 of genes required for cell survival in a mechanism much like miRNA-induced silencing. We statement that this most harmful 6mer seeds are G-rich with a G enrichment towards 5 end targeting SGs with a high C content in their 3UTR in a miRNA-like manner. Many tumor-suppressive miRNAs such as miR-34a-5p but none of the established oncogenic miRNAs contain G-rich 6mer seeds and most of miR-34a-5p’s toxicity comes from its 6mer seed sequence. Mature miRNAs from older and more conserved miRNAs contain less toxic seeds. We demonstrate that for most miRNAs the more abundant mature form corresponds to the arm that contains the less harmful seed. In contrast, for major tumor-suppressive miRNAs, the mature miRNA is derived from the arm that harbors the more harmful seed. Our data allow.