The import of nuclear encoded tRNA and rRNA into the mitochondria has been demonstrated 
. This raised an interesting question of possible import of small non-coding RNAs including miRNA to the mitochondria, which may fine-tune the levels of target proteins associated with mitochondria and under various physiological conditions. In this study, we described the association of sRNAs including miRNA with mitochondria of two human cell lines HEK293 and HeLa through deep sequencing. The targets of identified known and putative novel miRNAs may regulate critical cellular pathways where mitochondria plays key role.
The deep sequencing of mitochondrial associated RNA confirmed the presence of diverse population of sRNAs: snRNA, srpRNA and snoRNA. The association of diverse population of sRNAs is intriguing and suggests either the role of mitochondria in their biogenesis or the role of sRNA in regulation of mitochondrial functions. The snRNA and snoRNA are transported back from cytoplasm to sub-nuclear regions 
. It has been observed that snoRNA are processed in a similar manner like miRNA 
and may perform functions similar to miRNA apart from its role in ribosomal biogenesis. The association of snoRNA with mitochondria needs to be further studied for their role in mitochondrial ribosome assembly, RNA modifications or miRNA like functions. The association of srpRNA also suggests that mitochondria may be involved in the translational of the mRNA encoding mitochondrial targeted proteins. These observations were further strengthened from the recent study describing the mitochondrial transcriptome where snRNA, snoRNA and srpRNA were observed to be associated with mitochondria 
. Interestingly, we also observed piRNA associated with mitochondria from both HEK293 and HeLa cells. It has been observed earlier that piRNAs are generally expressed in germ line tissues but emerging evidences suggest the presence of piRNA in different somatic tissue 
. The expression of piRNA was observed in 17 out of the 40 mouse somatic tissues and cell types. The piRNA expressed in somatic tissues showed characteristics of piRNA such as length distribution between 24 and 32 nt, 5′ uridine bias and clustering along the genome along with universal piRNA features. The association of piRNA with mitochondria of HEK293 and HeLa in this study suggests either mitochondria are involved in biogenesis of piRNA or postranscriptionally associates with mitochondria like miRNA. The role of mitochondria in biogenesis of piRNA has been further supported by recent studies. Two groups have clearly demonstrated the role of mitochondrial protein known as MitoPLD/zuc (Drosophila homologue) in piRNA biogenesis and piRNA mediated silencing both in fly and mouse germlines 
. This suggests that mitochondria may be an important player in piRNA pathways.
Interestingly, 2–5% sRNA reads was categorized to be miRNA supporting the recent evidence of existence of mitochondrial associated miRNA 
. During our manuscript preparation, mitochondrial transcriptome was analyzed 
which is in consonance with our study and observed that 3% of sRNAs showed similarity to miRNA. Interestingly, we observed that miRNA identified in recent studies 
as well as microarray of mitochondrial RNA from our lab (data not published) was found to be widely represented in our deep sequencing results. This strongly suggests that our deep sequencing approach confirmed the validated results observed in the previous studies. The further analysis showed that the frequency of association of 35 miRNAs with mitochondria of HEK293 and HeLa were similar whereas majority of miRNAs were differentially associated in both cell lines. Similarly, 196 and 13 putative novel miRNAs were associated with mitochondria from HEK293 and HeLa respectively. These evidences suggest that association of miRNAs with mitochondria may be cell type and stimulus specific which needs further analysis using different cell lines and in different conditions. Interestingly, base biasness of miRNA (22 nt) showed preference for U at the first position in both HEK293 and HeLa suggesting mitochondrial associated miRNA may have high affinity to preferentially bind to MID domain of Ago2 and may be involved in binding with targets for gene regulation 
. Further, it was observed that 4 known and 24 putative novel miRNA aligned to mitochondrial genome at different positions corresponding to 16S rRNA, tRNA and subunits of complex I. These miRNAs may be either mitochondrial genome encoded or nuclear encoded, may regulate mitochondrial transcripts involved in ribosome assembly and electron transport chain. This will require further experimental validations.
We identified the targets of miRNAs associated with mitochondria with high frequency count from both HEK293 and HeLa. Interestingly, GO terms related to RNA transcription were enriched. Ago2, core component of RISC complex involved in regulation and turnover of target mRNA has been reported to be colocalized with mitochondria 
. Our results also showed the association of Ago2 and Ago3 with mitochondria (Figure S3
). It has been well established that Ago proteins associates with miRNA 
. Interestingly, Ago1–3 has recently been demonstrated to be redundantly associated with diverse population of RNA apart from miRNA including tRNA, vRNA, snoRNA, rRNA pseudogenes, snRNA, tiRNA, TSSa-RNA, PASRs, etc 
which may be one of the reasons explaining the observed association of these RNAs to mitochondria. These sRNAs are known to regulate mRNA turnover and translation which again emphasizes the possible role of mitochondria in these processes. Similarly, it has been observed that mitochondria also associate with P bodies 
. These P bodies are cytoplasmic granules that are linked to mRNA decay, mRNA storage and RNA silencing. In our study, we also observed that miRNAs and target mRNAs associated with mitochondria were enriched to the outer membrane of mitochondria. This is also supported by recent study of mitochondrial transcriptome where depletion of outer membrane of mitochondria results in selective loss of miRNAs 
and other RNAs. These evidences strongly suggest that the outer membrane may assemble novel complexes by associating the proteins of RISC complexes, target mRNA to regulate site-specific turnover of mRNA and translation.
Analysis of putative novel miRNAs associated with mitochondria from HEK293 and HeLa showed enrichment of GO functions related to positive/negative regulation of RNA which has been discussed above that further supported our hypothesis. Interestingly, putative novel miRNAs identified in our study also showed enrichment of GO functions related to several signaling pathways important in cancer and metabolism where mitochondria plays crucial role. Apoptosis, cell cycle, kinase activity and proteins transport related pathways were enriched. Some of the known miRNA-target interactions have been demonstrated previously. hsa-miR-103/107 is highly associated with mitochondria (as observed in our study) which regulates cellular Acetyl-CoA and lipid levels 
. Similarly hsa-miR-181 targets multiple Bcl-2 family members and regulates apoptosis 
. These evidences suggest that miRNAs associate with mitochondria and regulates the turnover of mRNA and levels of protein that are implicated in mitochondrial related functions.
In conclusion, our study using subcellular fractionation and deep sequencing demonstrated the association of sRNAs including miRNA to mitochondria. The putative novel miRNAs needs further characterization for their function, association with mitochondria and translocation to other subcellular sites during different cellular processes. The association of miRNAs, their target mRNAs and Ago2/3 with mitochondria suggests that mitochondrial outer membrane may probably provide novel platform to assemble the miRNA/RISC complexes to regulate the subcellular site-specific protein levels. The further study in this direction will help to understand many unanticipated role of mitochondria and associated miRNA in different physiological and pathological conditions.