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MicroRNAs (miRNAs) post‐transcriptionally repress almost all genes in mammals and thereby form an additional layer of gene regulation. As such, miRNAs impact on nearly every physiological process and have also been associated with cancer. Prominent examples of such miRNAs can be found in the miR‐15 family, composed of the bicistronic clusters miR‐15a/16‐1, miR‐15b/16‐2, and miR‐497/195. In particular, the miR‐15a/16‐1 cluster is deleted in almost two thirds of all chronic B lymphocytic leukemia (CLL) cases, a phenotype that is also recapitulated by miR‐15a/16‐1‐deficient as well as miR‐15b/16‐2‐deficient mice. Under physiological conditions, those two clusters have been implicated in T‐cell function, and B‐cell and natural killer (NK) cell development; however, it is unclear whether miR‐497 and miR‐195 confer similar roles in health and disease. Here, we have generated a conditional mouse model for tissue‐specific deletion of miR‐497 and miR‐195. While mice lacking miR‐15a/16‐1 in the hematopoietic compartment developed clear signs of CLL over time, aging mice deficient for miR‐497/195 did not show such a phenotype. Likewise, loss of miR‐15a/16‐1 impaired NK and early B‐cell development, whereas miR‐497/195 was dispensable for these processes. In fact, a detailed analysis of miR‐497/195‐deficient mice did not reveal any effect on steady‐state hematopoiesis or immune cell function. Unexpectedly, even whole‐body deletion of the cluster was well‐tolerated and had no obvious impact on embryonic development or healthy life span. Therefore, we postulate that the miR‐497/195 cluster is redundant to its paralog clusters or that its functional relevance is restricted to certain physiological and pathological conditions., The miR‐15a/16‐1 and miR‐15b/16‐2 clusters regulate hematopoiesis and are key tumor suppressors in chronic lymphocytic leukemia. However, little is known about the third cluster of the miR‐15 family, miR‐497/195. Here, we show that miR‐497/195 is dispensable for immune cell development and function, and that its loss does not provoke any overt malignancies. This suggests a redundant role for miR‐497/195 or that its functional relevance is restricted to certain conditions.

SummaryMicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally silence most protein-coding genes in mammals. They are generated from primary transcripts containing single or multiple clustered stem-loop structures that are thought to be recognized and cleaved by the DGCR8/DROSHA Microprocessor complex as independent units. Contrasting this view, we here report an unexpected mode of processing of a bicistronic cluster of the miR-15 family, miR-15a-16-1. We find that the primary miR-15a stem-loop is a poor Microprocessor substrate and is consequently not processed on its own, but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage, and describe SAFB2 as a novel accessory protein of DROSHA. Notably, SAFB2-mediated cluster assistance expands to other clustered pri-miRNAs including miR-15b, miR-92a and miR-181b, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal DGCR8/DROSHA substrates in clustered primary miRNA transcripts.Highlightsthe primary miR-15a stem-loop structure per se is a poor Microprocessor substratecleavage of pri-miR-15a requires the processing of an additional miRNA stem-loop on the same RNAsequential pri-miRNA processing or “cluster assistance” is mediated by SAFB proteinsSAFB2 associates with the Microprocessor

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.