To our knowledge, no approach for targeted sequencing of mitochondrial RNA is available until now. To address this demand, we have developed an optimized approach specifically suitable for targeted sequencing of whole mitochondrial transcriptome in a broad range of sample types. Our novel approach has several advantages: (i) it provides significant relevance to the selection of optimal experimental parameters for mtRNA sequencing by integrating the atypical features of mitochondrial transcription; (ii) it is competitive in economic terms, allowing comprehensively analysis of mitochondrial transcriptome with satisfactory resolution but reduced cost; (iii) it provides a powerful tool to investigate the hierarchy of mitochondrial transcription initiation and processing and thus enables a broad-spectrum characterization of mitochondrial transcriptome; (iv) it confers widespread applicability in the detection of different types of specimens, including plasma and single-cell samples.
Broader applications of RNA seq have greatly transformed our understanding on human nuclear transcriptome and revealed many essential phenotype-related features, including differential gene expression, alternative RNA splicing and modification (Bradley and Anczuków, 2023), (de Klerk and t Hoen, 2015). Compared to the nucleus, the mitochondria have evolved its unique features with respect to the organization and regulation of gene transcription (Jensen et al., 1988), whereas optimal RNA seq specifically for better characterization of mtRNAs is still lacking. In the present study, we comprehensively evaluated the influence of essential experimental protocols, including library construction, RNA enrichment, rRNA depletion, on accurately profiling mitochondrial transcriptome in WTS data. Our results indicate that stranded RNA-seq provides a more accurate identification of mitochondrial transcripts derived from either H or L strand when compared with non-stranded RNA-seq and is therefore recommended for mtRNA sequencing. In addition, the polyadenylation of mRNAs is critical for the stability and translation of transcripts (Gruber and Zavolan, 2019). A series of previous studies have reported that the polyadenylation is essential for completion of stop codons in 7 of the 13 H strand mt-mRNAs (ND1-4, CTYB, COX3 and ATP6) and the L strand mt-mRNA (ND6) is not polyadenylated (Rackham and Filipovska, 2022) (Pearce et al., 2017). Therefore, considering the different levels of mt-RNA polyadenylation, we evaluated the feasibility of the two commonly used RNA enrichment approaches, polyA selection and rRNA depletion, in mtRNA sequencing and found that the rRNA depletion was indeed a more suitable approach for the accurate profiling of mtRNAs. Furthermore, the 0.1X rRNA probe was demonstrated to retain a relative low level of mt-rRNAs while not alter the original mt-rRNA profile and was thus selected as optimal rRNA depletion in tissue samples, whereas further optimization is needed for other sample types, such as plasma. Based on these insights of performance evaluations, the highly efficient protocols can be selected for the capture-based mtRNA seq.
Although emerging WTS-based studies have raised great promise for diagnosis and treatment of various diseases, low-level mtRNAs may be unannotated under current protocols due to the insufficient coverage of mtRNAs. Recent studies have demonstrated the feasibility of capture-based approach in obtaining the enriched read coverage and accurate quantitation for nuclear RNA (Bussotti et al., 2016), (Mercer et al., 2011a), whereas the approach for mtRNA target enrichment and sequencing is still lacking till now. Hence, in the present study, we attempted to develop a highly efficient approach to interrogate mtRNA in a manner with lower costs and unprecedented resolution. Compared with WTS-based approach, capture-based NGS approach has a great decrease in required data volume, without affecting the sensitivity and accuracy of detection. In addition, the capture-based approach also characterized the unannotated mt-tRNA transcripts whose expression level is below the detection limits of conventional WTS. As a proof-of-concept characterization of mtRNAs, the transcription initiation and pausing sites were identified based on capture-based mtRNA seq. Additionally, due to the polycistronic nature of mitochondrial transcription, post-transcriptional events are pivotal in determining downstream events. We hence identified putative mtRNA cleavage sites and ratios by capture-based mtRNA seq, which may contribute to elaborate key mtRNA processing events. Moreover, the capture-based mtRNA seq also had a very reliable performance in plasma and single-cell samples, highlighting its wide application. Together, the present study has established a highly efficient mtRNA targeted sequencing approach, which enables unbiased mtRNA detection, showing evenly captured transcripts across mitochondrial transcriptome and therefore maintaining the original features of transcription.
Despite the above advantages, the capture-based mtRNA seq still needs to be optimized in several respects. First, the designed capture probes were fragmented products of mtDNA PCR amplification, which may be insufficient when studying novel or rare mtRNA intermediates. Nevertheless, the unnecessarily exact complementary matches between probes and target cDNAs together with the high depth coverage may serve as a versatile solution to comprehensively detect various intermediates and single-nucleotide polymorphisms in mtRNAs. Second, several technical defects may limit the completeness of capture-based NGS approach, including the shorter sequencing reads than many transcripts, incomplete reverse transcription of RNA templates, and RNA degradation before reverse transcription. These issues should be taken into consideration in the improvements of future protocols. The combination of third-generation sequencing and template-switching approaches would be appreciated.
To the best of our knowledge, this is the first study to provide a highly efficient capture-based mtRNA sequencing approach, holding great promise for the comprehensive analysis of human mitochondrial transcriptome that is far from fully characterized. We anticipate that based on the established approach in our study, the previously unknown features of mitochondrial transcription (such as initiation, processing, maturation and degradation of mtRNAs) would be efficiently delineated, perhaps adding more complementary views beyond previous WTS-based studies. Thus, our approach may pave the path toward functional mtRNA annotations, and helps to explore more extraordinary complexity of the mitochondrial transcriptional landscape.