Widespread association of the Argonaute protein AGO2 with meiotic chromatin suggests a distinct nuclear function in mammalian male reproduction

Abstract

Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and although its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo using spermatogenic cells as a model. We find that AGO2 transiently binds both chromatin and nucleus-specific mRNA transcripts of hundreds of genes required for sperm production during male meiosis in mice, and that germline conditional knockout (cKO) of Ago2 causes depletion of the encoded proteins. Correspondingly, Ago2 cKO males show abnormal sperm head morphology and reduced sperm count, along with reduced postnatal viability of offspring. Together, our data reveal an unexpected nuclear role for AGO2 in enhancing expression of developmentally important genes during mammalian male reproduction.

Deconvolution of in vivo protein-RNA contacts using fractionated eCLIP-seq

“…To apply our pipeline without membrane size fractionation, cut a lane from the protein size to approximately 80 kD above it. For AGO2 eCLIP-seq ( Griffin et al, 2022 ), according to our western blot results and to the signal on the developed film, we cut a lane starting between 75-100 kD (AGO2 size is 97 kD) and ending approximately at 250 kD ( Figure 1 bottom panel). Note: Control samples should show absent or significantly reduced signal in comparison to experimental samples ( Figure S1 ), resulting in insufficient material for library preparation and sequencing and therefore confirming insignificant non-specific binding.…”

“…To apply our pipeline without membrane size fractionation, cut a lane from the protein size to approximately 80 kD above it. For AGO2 eCLIP-seq ( Griffin et al, 2022 ), according to our western blot results and to the signal on the developed film, we cut a lane starting between 75-100 kD (AGO2 size is 97 kD) and ending approximately at 250 kD ( Figure 1 bottom panel).…”

“…FreCLIP-seq is based on previously published iCLIP-seq and eCLIP-seq protocols ( Hafner et al., 2021 ; König et al., 2010 ; Van Nostrand et al., 2016 ), with the introduction of the membrane size fractionation step that allows to separate RNA binding signals of individual proteins within a complex. The experimental protocol described here has been successfully applied with and without membrane size fractionation in several cellular models, including inducible and standard cell lines for U2AF1 and U2AF2 binding analysis ( Biancon et al., 2022 ; Glasser et al., 2022 ), and cytoplasmic fractions derived from primary mouse cells for AGO2 binding analysis ( Griffin et al, 2022 ).…”

“…(2022) Griffin et al (2022) GEO: GSE195620 ; GSE181264 freCLIP-seq and eCLIP-seq analysis code Biancon et al. (2022) This paper GitHub: https://github.com/TebaldiLab/eCLIP_seq https://doi.org/10.5281/zenodo.7121228 Original Images This paper Mendeley Data: https://doi.org/10.17632/m7g92hkwmh.1 Experimental models: Cell lines Human: HEL ATCC TIB-180 Experimental models: Organisms/strains Mouse: Ago2 conditional knockout males (Ddx4-Cre/+;Ago2 flox/Δ ) Griffin et al (2022) N/A Oligonucleotides RNA oligos (X1A/B, X2A/B, RiL19) and DNA oligos (AR17, rand3Tr3) Van Nostrand et al. (2016) eCLIP Standard Operating Procedure v1.P 20151108 Illumina Adapters (D501-508, D701-D712) Illumina Adapter Sequences https://support-docs.illumina.com/SHARE/AdapterSeq/illumina-adapter-sequences.pdf Pages 50–51.…”

“…An implementation of these steps is available as bash and R scripts ( https://github.com/TebaldiLab/eCLIP_seq ). The case study shown is based on a dataset where AGO2 binding to cytoplasmic RNAs was quantified in mouse whole testis cells ( Griffin et al, 2022 ). For any step, alternative computational tools can be used, with additional care optionally necessary to optimize the compatibility of input/output file formats.…”