Periodic Reporting for period 1 - EpiSperm (Dissecting the role of sperm transcriptome dynamics in intergenerational inheritance through native RNA nanopore sequencing)
Période du rapport: 2022-10-01 au 2025-03-31
Mammalian sperm RNA is increasingly recognized as an additional source of paternal hereditary information beyond DNA. Environmental inputs, such as diet and stress, can reshape the sperm RNA signature and induce offspring phenotypes that relate to paternal environmental stressors. However, how, when and to what extent sperm RNA populations change, and what is the role that RNA modifications and other post-transcriptional regulatory layers play in shaping sperm RNA dynamics, remains poorly understood.
Here, we propose to characterize the dynamics of RNA populations during sperm formation and maturation using native RNA nanopore sequencing. This technology is suited to provide an integrative and comprehensive view of the transcriptome, epitranscriptome, degradation patterns and tailing dynamics simultaneously, and with single molecule resolution. We will establish novel library preparation methods that can capture the full sperm (epi)transcriptome, and will capitalize on our recently developed algorithms to map and quantify RNA modifications in individual RNA molecules. We will then apply these methods to reveal how paternal dietary exposures affect sperm RNA populations and the metabolic phenotypes of their offspring, and test whether the novel identified RNA candidates can transmit diet-induced paternal phenotypes to the subsequent generation. The integrative nature of our approach, which combines transcriptomics, epitranscriptomics, fragmentomics and tailomics, has the potential to provide answers to how environmental traits are inherited across generations, as well as reveal previously unidentified carriers of intergenerational inheritance, which we will experimentally test and orthogonally validate as part of this proposal. Finally, we propose to expand our previous work on direct RNA multiplexing to establish single cell direct RNA nanopore sequencing, to characterize the diversity and heterogeneity of the sperm RNA (epi)transcriptome at an unprecedented single cell and single molecule resolution. This technology will allow us not only to examine the sperm epitranscriptome and its heterogeneity at an unprecedented resolution, but will also have a major impact in many other scientific disciplines, such as cancer therapies. Moreover, the possibility of studying the epitranscriptome at single cell and single molecule resolution will greatly facilitate the functional dissection of the many RNA modifying enzymes whose functions are yet to be elucidated, but are known to be the cause of diverse human diseases when dysregulated.
With EPISPERM, we propose to obtain a comprehensive and integrative multi-omics view (transcriptomics, epitranscriptomics, fragmentomics and tailomics) of the dynamics of sperm RNA across maturation stages (Aim 1), upon different diets (Aim 2) and across individual cells (Aim 3). These aims will include experiments to functionally test whether identified RNA modifications and RNA biotypes play a role in sperm maturation and in intergenerational inheritance.
Here, we propose to characterize the dynamics of RNA populations during sperm formation and maturation using native RNA nanopore sequencing. This technology is suited to provide an integrative and comprehensive view of the transcriptome, epitranscriptome, degradation patterns and tailing dynamics simultaneously, and with single molecule resolution. We will establish novel library preparation methods that can capture the full sperm (epi)transcriptome, and will capitalize on our recently developed algorithms to map and quantify RNA modifications in individual RNA molecules. We will then apply these methods to reveal how paternal dietary exposures affect sperm RNA populations and the metabolic phenotypes of their offspring, and test whether the novel identified RNA candidates can transmit diet-induced paternal phenotypes to the subsequent generation. The integrative nature of our approach, which combines transcriptomics, epitranscriptomics, fragmentomics and tailomics, has the potential to provide answers to how environmental traits are inherited across generations, as well as reveal previously unidentified carriers of intergenerational inheritance, which we will experimentally test and orthogonally validate as part of this proposal. Finally, we propose to expand our previous work on direct RNA multiplexing to establish single cell direct RNA nanopore sequencing, to characterize the diversity and heterogeneity of the sperm RNA (epi)transcriptome at an unprecedented single cell and single molecule resolution. This technology will allow us not only to examine the sperm epitranscriptome and its heterogeneity at an unprecedented resolution, but will also have a major impact in many other scientific disciplines, such as cancer therapies. Moreover, the possibility of studying the epitranscriptome at single cell and single molecule resolution will greatly facilitate the functional dissection of the many RNA modifying enzymes whose functions are yet to be elucidated, but are known to be the cause of diverse human diseases when dysregulated.
With EPISPERM, we propose to obtain a comprehensive and integrative multi-omics view (transcriptomics, epitranscriptomics, fragmentomics and tailomics) of the dynamics of sperm RNA across maturation stages (Aim 1), upon different diets (Aim 2) and across individual cells (Aim 3). These aims will include experiments to functionally test whether identified RNA modifications and RNA biotypes play a role in sperm maturation and in intergenerational inheritance.
* Improvement of direct RNA sequencing library preparation to capture short RNA fragments, such as tRNAs
* Development of novel protocols to capture coding and non-coding transcriptome including tail length estimation and tail heterogeneity, with isoform resolution
* Establishment of HFD as paradigm of intergenerational inheritance, measured in the form of GTT and ITT tests.
* Experimental setup to examine the reversibility of intergenerational inheritance phenotypes, in the form of diet reversal, with different durations.
* Establishment of FACS-sorted spermatogenic cells from mice subjected to different diet combinations
* Establishment of sperm RNA extraction without somatic cell contamination
* Establishment of sperm direct RNA sequencing protocol and application to HFD and SFD-fed mice
* Bioinformatic pipeline to analyze sperm RNA datasets generated using direct RNA sequencing
* Orthogonal validation of results using LC-MS/MS and Illumina short-read sequencing
* Analysis of transcriptomic changes in F1 generation, to identify key pathways dysregulated as an effect of paternal diet
* Establishment of novel multiplexing strategy for direct RNA sequencing, and demultiplexing algorithm, up to 96 barcodes
* Development of novel approach to demultiplex extended barcodes for future single cell sequencing experiments
* Development of novel protocols to capture coding and non-coding transcriptome including tail length estimation and tail heterogeneity, with isoform resolution
* Establishment of HFD as paradigm of intergenerational inheritance, measured in the form of GTT and ITT tests.
* Experimental setup to examine the reversibility of intergenerational inheritance phenotypes, in the form of diet reversal, with different durations.
* Establishment of FACS-sorted spermatogenic cells from mice subjected to different diet combinations
* Establishment of sperm RNA extraction without somatic cell contamination
* Establishment of sperm direct RNA sequencing protocol and application to HFD and SFD-fed mice
* Bioinformatic pipeline to analyze sperm RNA datasets generated using direct RNA sequencing
* Orthogonal validation of results using LC-MS/MS and Illumina short-read sequencing
* Analysis of transcriptomic changes in F1 generation, to identify key pathways dysregulated as an effect of paternal diet
* Establishment of novel multiplexing strategy for direct RNA sequencing, and demultiplexing algorithm, up to 96 barcodes
* Development of novel approach to demultiplex extended barcodes for future single cell sequencing experiments
* We have developed a novel approach to capture short RNAs using direct RNA sequencing library preparation, focused on tRNAs
RESULTS: Lucas et al, Nat Biotech 2024; patent filed and licensed
* We have developed a novel protocols to capture coding and non-coding transcriptome including tail length estimation and tail heterogeneity, with isoform resolution
RESULTS: Begik et al., Nat Methods 2023; Begik et al., bioRxiv 2024
* We have established and performed sperm direct RNA sequencing protocol and application to HFD and SFD-fed mice, including bioinformatic pipeline to analyze sperm RNA datasets
RESULTS: method established, and samples sequenced for first combination of diets (HFD14 and SFD14). Future work to potentially improve the method, ongoing work on data analysis and interpretation of results
* We have established a novel multiplexing strategy for direct RNA sequencing, and demultiplexing algorithm, up to 96 barcodes, and are currently development a novel approach extend this work to increased number of barcodes for future single cell sequencing experiments
RESULTS: Pryszcz et al., bioRxiv 2024, 2 patents filed.
RESULTS: Lucas et al, Nat Biotech 2024; patent filed and licensed
* We have developed a novel protocols to capture coding and non-coding transcriptome including tail length estimation and tail heterogeneity, with isoform resolution
RESULTS: Begik et al., Nat Methods 2023; Begik et al., bioRxiv 2024
* We have established and performed sperm direct RNA sequencing protocol and application to HFD and SFD-fed mice, including bioinformatic pipeline to analyze sperm RNA datasets
RESULTS: method established, and samples sequenced for first combination of diets (HFD14 and SFD14). Future work to potentially improve the method, ongoing work on data analysis and interpretation of results
* We have established a novel multiplexing strategy for direct RNA sequencing, and demultiplexing algorithm, up to 96 barcodes, and are currently development a novel approach extend this work to increased number of barcodes for future single cell sequencing experiments
RESULTS: Pryszcz et al., bioRxiv 2024, 2 patents filed.