DAZL mediates a broad translational program regulating expansion and differentiation of spermatogonial progenitors

Abstract

Fertility across metazoa requires the germline-specific DAZ family of RNA-binding proteins. Here we examine whether DAZL directly regulates progenitor spermatogonia using a conditional genetic mouse model and in vivo biochemical approaches combined with chemical synchronization of spermatogenesis. We find that the absence of Dazl impairs both expansion and differentiation of the spermatogonial progenitor population. In undifferentiated spermatogonia, DAZL binds the 3' UTRs of ~2,500 protein-coding genes. Some targets are known regulators of spermatogonial proliferation and differentiation while others are broadly expressed, dosage-sensitive factors that control transcription and RNA metabolism. DAZL binds 3' UTR sites conserved across vertebrates at a UGUU(U/A) motif. By assessing ribosome occupancy in undifferentiated spermatogonia, we find that DAZL increases translation of its targets. In total, DAZL orchestrates a broad translational program that amplifies protein levels of key spermatogonial and gene regulatory factors to promote the expansion and differentiation of progenitor spermatogonia.

Whole-Transcriptome Sequencing-Based Analysis of DAZL and Its Interacting Genes during Germ Cells Specification and Zygotic Genome Activation in Chickens

“…Notably, the UGUU(U/A) motif is identified in most predicted DAZL interacting genes. This motif is recently reported as the most preferential motif for DAZL binding than all other motifs mentioned above [ 17 ]. Moreover, the longer 3’UTRs contained more counts, and the shorter 3’UTRs contained fewer counts of the DAZL binding motifs.…”

“…Therefore, we analyzed the DAZL interacting genes in this study based on the conserved DAZL binding motifs reported in other species. In the results, we identified different counts of the reported DAZL binding motifs, such as UGUU(U/A) [ 17 ], UGUU [ 16 ], GUU(U/A) [ 17 ], GUUG [ 13 ], GUUC [ 26 ], and UUU(C/G)UUU [ 15 , 27 ], in the 3’UTR of the predicted DAZL interacting genes in chicken ( Table S1 and Figure S1 ). Notably, the UGUU(U/A) motif is identified in most predicted DAZL interacting genes.…”

“…DAZL protein binds with thousands of mRNA transcripts at the 3’UTR, as reported in different species. In undifferentiated mouse spermatogonia, DAZL binds with about 2500 transcripts at a UGUU(U/A) motif and increases the translation of key spermatogonial gene regulatory factors [ 17 ]. In the mouse testis, DAZL binds with about 3000 transcripts, particularly at a UGUU motif, and increases the translation of binding mRNAs associated with the spermatogenesis [ 16 ].…”

“…The 3’ UTR sequences of DAZL interacting genes are extracted based on the coordinates of the stop codon and the end position of each transcript defined by the galGal6a reference genome (RefSeq assembly accession: GCF_000002315.6) and the corresponding generic feature format (GFF) gene annotation available under the BioProject PRJNA13342. The previously reported DAZL protein binding motifs, such as UGUU(U/A) [ 17 ], UGUU [ 16 ], GUU(U/A) [ 17 ], GUUC [ 26 ], GUUG [ 13 ], and UUU(C/G)UUU [ 15 , 27 ], are investigated by screening the 5’ to 3’ direction of the 3’ UTR sequences of each transcript on + or − strand via an in-house Python script (Python 3.7.3). Using a sliding window sequence of 4, 5, and 7 bp in length from the 3’ UTR, the occurrence of a perfect match to the above binding motifs is counted.…”

“…Due to its RNA-binding properties, DAZL protein binds with the 3′ untranslated region (UTR) of target mRNAs at the GUU-enriched regions [ 13 ]. DAZL enhances the translation of genes critically required for normal functioning of germ cells at various stages, including germ cells specification, development, and differentiation [ 13 , 14 , 15 , 16 , 17 ]. In addition, DAZL acts as a translational repressor of core pluripotency-, somatic differentiation-, and apoptosis-related genes in nascent PGCs [ 14 ], protecting its survivability and ability to differentiate into germline cells (spermatogonia/oogonia).…”