Historical RNA expression profiles from the extinct Tasmanian tiger

Abstract

Palaeogenomics continues to yield valuable insights into the evolution, population dynamics, and ecology of our ancestors and other extinct species. However, DNA sequencing cannot reveal tissue-specific gene expression, cellular identity, or gene regulation, only attainable at the transcriptional level. Pioneering studies have shown that useful RNA can be extracted from ancient specimens preserved in permafrost and historical skins from extant canids, but no attempts have been made so far on extinct species. We extract, sequence and analyze historical RNA from muscle and skin tissue of a ~130-year-old Tasmanian tiger (Thylacinus cynocephalus) preserved in desiccation at room temperature in a museum collection. The transcriptional profiles closely resemble those of extant species, revealing specific anatomical features such as slow muscle fibers or blood infiltration. Metatranscriptomic analysis, RNA damage, tissue-specific RNA profiles, and expression hotspots genome-wide further confirm the thylacine origin of the sequences. RNA sequences are used to improve protein-coding and noncoding annotations, evidencing missing exonic loci and the location of ribosomal RNA genes, while increasing the number of annotated thylacine microRNAs from 62 to 325. We discover a thylacine-specific microRNA isoform that could not have been confirmed without RNA evidence. Finally, we detect traces of RNA viruses, suggesting the possibility of profiling viral evolution. Our results represent the first successful attempt to obtain transcriptional profiles from an extinct animal species, providing thought-to-be-lost information on gene expression dynamics. These findings hold promising implications for the study of RNA molecules across the vast collections of Natural History museums and from well-preserved permafrost remains.

Transcriptome data from silica-preserved leaf tissue reveal gene flow patterns in a Caribbean bromeliad

“…The field of historical RNA is in its infancy. The recent sequencing of the Tasmanian tiger transcriptome has demonstrated that expression profiles can be obtained from museum collections ( Mármol-Sánchez et al , 2023 ). Historical RNA provides a new source of gene expression data, provides new data for microRNA prediction and enables superior genome annotation of rare or extinct species.…”

“…Unlike other approaches to analysis of population structure, conStruct incorporates spatial information into the analysis. A complementary approach to analysing and visualizing isolation by distance was conducted using FEEMS, which complements conStruct because it is robust to sparse sampling and functions well under simulated coalescent processes ( Marcus et al , 2021 ). FEEMS determines whether the amount of genetic distance between individuals is what would be expected based on geographical distance.…”

“…A fast estimation of effective migration surfaces (FEEMS) ( Marcus et al , 2021 ) analysis was performed to infer and visualize potential barriers to gene flow. The same set of SNPs was used for this analysis as for the conStruct analysis.…”

“…The recent emergence of the historical RNA field has brought forth the possibility that intact RNA might be obtained from unpreserved tissue. In metazoans, transcriptomic sequences have been obtained from a 130-year-old Tasmanian tiger ( Mármol-Sánchez et al , 2023 ). RNA allows for superior genome annotation, identification of microRNAs and analyses of transcriptomic profiles, making it invaluable for understanding genome evolution.…”

Temporal collections to study invasion biology

“…Further, epigenetic modifications can mediate environmental effects on phenotypes through gene expression and historical epigenomics may therefore allow ancestral patterns of gene expression to be inferred (e.g., Hahn et al., 2023; Rubi et al., 2020). These analyses could be coupled with direct reconstruction of gene expression profiles from natural history specimens (e.g., Marmol‐Sanchez et al., 2023), which is now possible, thanks to the development of protocols for extracting RNA from preserved specimens (e.g., Speer et al., 2022). DNA methylation patterns have already been investigated in ancient and historical specimens (e.g., Gokhman et al., 2014; Gokhman et al., 2016; Hahn et al., 2020; Niiranen et al., 2022; Orlando et al., 2015; Pedersen et al., 2014; Rubi et al., 2020; Smith et al., 2014), indicating that this approach holds promise for unravelling the role of heritable epigenetic modifications in facilitating rapid adaptation to environmental shifts.…”