12/30/2023 0 Comments Processsing pmouse and mousexHowever, the conventional ribosome profiling approach involves multiple steps with substantial loss of input material, restricting its application to samples with large numbers of cells. Transcriptome-wide mRNA translation can be measured by high-throughput sequencing of RNA fragments protected by ribosomes from nuclease digestion 9, 10. In particular, translational control of specific transcripts is essential for oocyte maturation and the oocyte-to-embryo transition 7, 8. Consequently, RNA expression and protein abundance are only modestly correlated until the late morula stage, emphasizing the need to elucidate post-transcriptional regulation during initial stages of embryogenesis 1, 5, 6. The early gene expression landscape is shaped by post-transcriptional regulation of maternal transcripts due to the absence of transcription from later stages of oocyte maturation through the early divisions of the embryo 3, 4. The Ribo-ITP approach will enable numerous applications by providing high-coverage and high-resolution ribosome occupancy measurements from ultra-low input samples including single cells. By integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle-stage oocytes is the predominant determinant of protein abundance in the zygote. These led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes and reduced translation efficiency of transcripts exhibiting allele-biased expression. Our high-coverage measurements enabled, to our knowledge, the first analysis of allele-specific ribosome engagement in early development. We identified differential translation efficiency as a key mechanism regulating genes involved in centrosome organization and N 6-methyladenosine modification of RNAs. Here, to address this challenge, we developed a novel microfluidic isotachophoresis (ITP) approach, named RIBOsome profiling via ITP (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. However, previous studies had been restricted to bulk measurements 2, precluding precise determination of translation regulation including allele-specific analyses. Translation regulation is critical for early mammalian embryonic development 1.
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