Analyses of RNA dynamics in Mus musculus

Ribonucleic acid (RNA) is a biological molecule that exists in the cell of virtually all kinds of known living organisms. Although its significance in fundamental cellular processes such as protein synthesis has been known for decades, an increasing number of novel species and functions of RNA have been discovered recently, and a complete picture of RNA functions in the cell is still elusive. One of the major challenges in RNA studies is that there had been no efficient method to quantify RNA with spatial and temporal information in vivo. In my doctoral studies, I developed a novel RNA sequencing method that metabolically labels RNA in a cell- and time-specific manner in mice and applied this method to solve biological problems that had been difficult to tackle. First, the robustness of the newly developed in vivo RNA labelling method was assessed. To confirm the sensitivity and specificity of RNA labelling, multiple transgenic mice that label RNA in different cell types were generated. By comparing the data obtained from each transgenic strain to previously generated transcriptomic datasets, I confirmed that RNA labelling in a specific cell type was achieved in all the strains analysed. This method would be useful to study cell-type-specific transcriptomics rather than the commonly used laborious and time-intensive cell isolation method often used, and might provide data that closely reflect the native transcriptional state in vivo. Next, using the same RNA labelling method, I tested if there is any RNA that is mobile between different cell types in mice. Intercellular mobility of RNA has been shown in nematodes and plants, but whether there is any RNA that is mobile between different mammalian cells in vivo is still unclear. The cell-specific RNA labelling method allowed us to assess the mobility of RNA directly for the first time. Based on previous publications, three different cell types were chosen as potential “donor” cells, and transgenic mice that label RNA in these cells were generated. The donor cell-derived labelled RNA was sought in “recipient” tissues that are not capable of labelling RNA. However, although RNA labelling was achieved in the donor tissues, no labelled RNA was found in the recipient tissues in any of the animals tested. This experiment presents a novel methodology to assess the mobility of RNA in living mice, and the obtained data suggest that only minor intercellular transfer of RNA, at best, is happening in the tested pairs of tissues. In the final part of my thesis, I applied the metabolic RNA labelling method to study the transcriptional dynamics in the early preimplantation mouse embryos. Unlike conventional RNA sequencing methods that can only quantify RNA abundance in each stage of the embryos, metabolic RNA sequencing can directly interrogate the transcriptional activity of each gene. This method is particularly powerful in studying the transcriptional network in the early preimplantation embryo, where embryo-derived RNA needs to be distinguished from maternally-deposited RNA. By exposing the mouse embryos to a nucleotide analogue in a stage-specific manner, I identified genes that are actively transcribed in the 2-cell embryos. This method would be useful in studying the transcriptional cascade in the early mammalian preimplantation embryos

Mature sperm small-RNA profile in the sparrow: implications for transgenerational effects of age on fitness.

Mammalian sperm RNA has recently received a lot of interest due to its involvement in epigenetic germline inheritance. Studies of epigenetic germline inheritance have shown that environmental exposures can induce effects in the offspring without altering the DNA sequence of germ cells. Most mechanistic studies were conducted in laboratory rodents and C.elegans while observational studies confirm the phenotypic phenomenon in wild populations of humans and other species including birds. Prominently, paternal age in house sparrows affects offspring fitness, yet the mechanism is unknown. This study provides a first reference of house sparrow sperm small RNA as an attempt to uncover their role in the transmission of the effects of paternal age on the offspring. In this small-scale pilot, we found no statistically significant differences between miRNA and tRNA fragments in aged and prime sparrow sperm. These results indicate a role of other epigenetic information carriers, such as distinct RNA classes, RNA modifications, DNA methylation and retained histones, and a clear necessity of future studies in wild populations.This work was supported by Cancer Research UK (C13474/A18583, C6946/A14492) and the Wellcome Trust (104640/Z/14/Z, 092096/Z/10/Z) through E.A.M. W.M. was funded by The Nakajima Foundation and St John’s College Benefactors’ Scholarship. K.G. received funding from the Swiss National Science Foundation advanced mobility fellowship. J.S. was supported by Imperial College London

Sequencing cell-type-specific transcriptomes with SLAM-ITseq.

Analysis of cell-type-specific transcriptomes is vital for understanding the biology of tissues and organs in the context of multicellular organisms. In this Protocol Extension, we combine a previously developed cell-type-specific metabolic RNA labeling method (thiouracil (TU) tagging) and a pipeline to detect the labeled transcripts by a novel RNA sequencing (RNA-seq) method, SLAMseq (thiol (SH)-linked alkylation for the metabolic sequencing of RNA). By injecting a uracil analog, 4-thiouracil, into transgenic mice that express cell-type-specific uracil phosphoribosyltransferase (UPRT), an enzyme required for 4-thiouracil incorporation into newly synthesized RNA, only cells expressing UPRT synthesize thiol-containing RNA. Total RNA isolated from a tissue of interest is then sequenced with SLAMseq, which introduces thymine to cytosine (T>C) conversions at the sites of the incorporated 4-thiouracil. The resulting sequencing reads are then mapped with the T>C-aware alignment software, SLAM-DUNK, which allows mapping of reads containing T>C mismatches. The number of T>C conversions per transcript is further analyzed to identify which transcripts are synthesized in the UPRT-expressing cells. Thus, our method, SLAM-ITseq (SLAMseq in tissue), enables cell-specific transcriptomics without laborious FACS-based cell sorting or biochemical isolation of the labeled transcripts used in TU tagging. In the murine tissues we assessed previously, this method identified ~5,000 genes that are expressed in a cell type of interest from the total RNA pool from the tissue. Any laboratory with access to a high-throughput sequencer and high-power computing can adapt this protocol with ease, and the entire pipeline can be completed in <5 d.This work was supported by grants from Cancer Research UK (C13474/A18583, C6946/A14492) and the Wellcome Trust (104640/Z/14/Z, 092096/Z/10/Z) to E.A.M.; and a grant from the European Research Council (ERC-StG-338252 miRLIFE) to S.L.A. The IMP is generously supported by Boehringer Ingelheim. W.M. was supported by the Nakajima Foundation and St John’s College Benefactors’ Scholarship. K.G. was supported by a Swiss National Foundation postdoc mobility fellowship

Ancestral genome reconstruction enhances transposable element annotation by identifying degenerate integrants

&lt;p&gt;Enhanced hg38 transposable element (TE) annotation and associated RepeatMasker outputs of hg38 chromosomes&nbsp;and reconstructed ancestral genomes (RAGs).&lt;/p&gt;&lt;ul&gt;&lt;li&gt;Homo_sapiens_augmented.bed: Enhanced TE annotation in hg38 based on multiple RAGs.&lt;/li&gt;&lt;li&gt;Homo_sapiens_RepeatMasker_out.tar.gz: RepeatMasker outputs of hg38 chromosomes.&lt;/li&gt;&lt;li&gt;*.fa.out: RepeatMasker outputs of RAGs.&lt;/li&gt;&lt;li&gt;halliftover.sh, merge.R, repeatmasker.sh, rm2bed.R: Scripts used to achieve enhanced TE annotation in hg38.&lt;/li&gt;&lt;li&gt;*_ucsc_enhanced.bed: Enhanced TE annotation on UCSC-provided annotation based on the Eutherian RAG.&lt;/li&gt;&lt;/ul&gt

SLAM-ITseq: sequencing cell type-specific transcriptomes without cell sorting.

Cell type-specific transcriptome analysis is an essential tool for understanding biological processes in which diverse types of cells are involved. Although cell isolation methods such as fluorescence-activated cell sorting (FACS) in combination with transcriptome analysis have widely been used so far, their time-consuming and harsh procedures limit their applications. Here, we report a novel in vivo metabolic RNA sequencing method, SLAM-ITseq, which metabolically labels RNA with 4-thiouracil in a specific cell type in vivo followed by detection through an RNA-seq-based method that specifically distinguishes the thiolated uridine by base conversion. This method has successfully identified the cell type-specific transcriptome in three different tissues: endothelial cells in brain, epithelial cells in intestine and adipocytes in white adipose tissue. As this method does not require isolation of cells or RNA prior to the transcriptomic analysis, SLAM-ITseq provides an easy yet accurate snapshot of the transcriptional state in vivo

Alterations in sperm long RNA contribute to the epigenetic inheritance of the effects of postnatal trauma

Psychiatric diseases have a strong heritable component known to not be restricted to DNA sequence-based genetic inheritance alone but to also involve epigenetic factors in germ cells. Initial evidence suggested that sperm RNA is causally linked to the transmission of symptoms induced by traumatic experiences. Here, we show that alterations in long RNA in sperm contribute to the inheritance of specific trauma symptoms. Injection of long RNA fraction from sperm of males exposed to postnatal trauma recapitulates the effects on food intake, glucose response to insulin and risk-taking in adulthood whereas the small RNA fraction alters body weight and behavioural despair. Alterations in long RNA are maintained after fertilization, suggesting a direct link between sperm and embryo RNA.ISSN:1359-4184ISSN:1476-557