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Background The presence of nuclear mitochondrial DNA (numtDNA) has been reported within several nuclear genomes. Next to mitochondrial protein-coding genes, numtDNA sequences also encode for mitochondrial tRNA genes. However, the biological roles of numtDNA remain elusive. Results Employing in silico analysis, we identify 281 mitochondrial tRNA homologs in the human genome, which we term nimtRNAs (nuclear intronic mitochondrial-derived tRNAs), being contained within introns of 76 nuclear host genes. Despite base changes in nimtRNAs when compared to their mtRNA homologs, a canonical tRNA cloverleaf structure is maintained. To address potential functions of intronic nimtRNAs, we insert them into introns of constitutive and alternative splicing reporters and demonstrate that nimtRNAs promote pre-mRNA splicing, dependent on the number and positioning of nimtRNA genes and splice site recognition efficiency. A mutational analysis reveals that the nimtRNA cloverleaf structure is required for the observed splicing increase. Utilizing a CRISPR/Cas9 approach, we show that a partial deletion of a single endogenous nimtRNA Lys within intron 28 of the PPFIBP1 gene decreases inclusion of the downstream-located exon 29 of the PPFIBP1 mRNA. By employing a pull-down approach followed by mass spectrometry, a 3′-splice site-associated protein network is identified, including KHDRBS1, which we show directly interacts with nimtRNA Tyr by an electrophoretic mobility shift assay. Conclusions We propose that nimtRNAs, along with associated protein factors, can act as a novel class of intronic splicing regulatory elements in the human genome by participating in the regulation of splicing.

MicroRNAs (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.