|
| Status |
Public on Sep 27, 2022 |
| Title |
The NMD endonuclease SMG6 co-operates with the piRNA pathway in germ granules to shape the male germ cell transcriptome |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
Non-coding RNA profiling by high throughput sequencing
|
| Summary |
Nonsense-mediated RNA decay (NMD) is a conserved RNA turnover pathway. Here we report that the sole endonuclease in the NMD pathway, SMG6, is essential for the male germline. Germ-cell conditional knockout (cKO) of Smg6 causes complete arrest of spermatogenesis at the early haploid cell stage. Smg6-cKO round spermatids accumulate NMD target mRNAs with long 3’ untranslated regions (UTRs) and fail to eliminate transcripts normally expressed during meiosis, the previous step in spermatogenesis. SMG6 and PIWI-interacting (pi) RNA pathway components are highly enriched in the chromatoid body (CB), a specialized cytoplasmic granule in male germ cells. This led to the intriguing possibility that the CB is a site where SMG6 and the piRNA pathway co-operate to regulate RNA metabolism. Several findings supported this hypothesis: (i) SMG6 and the piRNA-binding protein PIWIL1 have almost identical temporal expression and localization patterns, (ii) SMG6 and PIWIL1 physically interact, (iii) scores of genes upregulated in Smg6-cKO round spermatids overlap with those upregulated in Piwil1-KO round spermatids, and (iv) the phenotypic defects caused by SMG6 loss phenocopies the defects caused by PIWIL1 loss. Together, our results demonstrate that SMG6 is an essential regulator of the male germline transcriptome and highlight the CB as a molecular platform coordinating RNA regulatory pathways to control sperm production and fertility.
|
| |
|
| Overall design |
A germ cell-specific Smg6 conditional knockout mouse line (Smg6-cKO) was generated by crossing a mouse line harboring loxP sites on either side of Smg6 exon 9 with a transgenic mouse line expressing the Cre recombinase under the control of the Neurogenin 3 (Ngn3)-promoter. Wild type-like littermates were used as controls. RNA sequencing was performed to analyze differential expression of long poly(A)-containing RNAs and small RNAs in pachytene spermatocytes (Spc) and round spermatids (RS), as well as differential levels of long RNAs in chromatoid bodies (CB). 1) RNA-seq of CBs: CBs were isolated from Smg6-cKO and control testes using anti-DDX4 immunoaffinity protocol. Four control samples and three Smg6-cKO samples were analyzed. For each sample, material from 2-4 individual mice were pooled. 2) RNA-seq and 3) smallRNA-seq of testicular cells: Both testes from either one adult Smg6-cKO or one adult control mouse were used to isolate RS and Spc using BSA-gradient velocity sedimentation. For RNA-seq, three biological replicates were analyzed per genotype. For smallRNA-seq, three RS and five Spc biological replicates were analyzed per genotype.
|
| |
|
| Contributor(s) |
Lehtiniemi T, Bourgery M, Ma L, Kotaja N |
| Citation(s) |
36259644 |
| |
| Submission date |
Aug 20, 2021 |
| Last update date |
Jan 03, 2023 |
| Contact name |
Matthieu Bourgery |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Turku
|
| Lab |
Noora Kotaja's lab
|
| Street address |
Kiinamyllynkatu 10
|
| City |
Turku |
| ZIP/Postal code |
FI-20520 |
| Country |
Finland |
| |
|
| Platforms (2) |
| GPL21493 |
Illumina HiSeq 3000 (Mus musculus) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
|
| Samples (35)
|
|
| Relations |
| BioProject |
PRJNA756554 |
| SRA |
SRP333479 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE182518_TEtranscripts_out_UCSC_CB_RNAseq.cntTable.txt.gz |
281.6 Kb |
(ftp)(http) |
TXT |
| GSE182518_TEtranscripts_out_UCSC_RS_RNAseq.cntTable.txt.gz |
315.1 Kb |
(ftp)(http) |
TXT |
| GSE182518_TEtranscripts_out_UCSC_Spc_RNAseq.cntTable.txt.gz |
318.5 Kb |
(ftp)(http) |
TXT |
| GSE182518_counts_piRNA_cluster_smallRNAseq.txt.gz |
15.9 Kb |
(ftp)(http) |
TXT |
| GSE182518_counts_piRNA_precursor_RS_RNAseq.txt.gz |
8.6 Kb |
(ftp)(http) |
TXT |
| GSE182518_counts_piRNA_precursor_Spc_RNAseq.txt.gz |
9.2 Kb |
(ftp)(http) |
TXT |
| GSE182518_counts_piRNA_precursors_CB_RNAseq.txt.gz |
9.5 Kb |
(ftp)(http) |
TXT |
| GSE182518_counts_piRNA_rmsk_smallRNAseq.txt.gz |
41.6 Kb |
(ftp)(http) |
TXT |
| GSE182518_mRNA_transcript_counts_ensembl_CB_RNAseq.txt.gz |
5.0 Mb |
(ftp)(http) |
TXT |
| GSE182518_mRNA_transcript_counts_ensembl_RS_RNAseq.txt.gz |
5.2 Mb |
(ftp)(http) |
TXT |
| GSE182518_mRNA_transcript_counts_ensembl_Spc_RNAseq.txt.gz |
5.2 Mb |
(ftp)(http) |
TXT |
| GSE182518_merge_miRNAs_smallRNAseq.txt.gz |
15.7 Kb |
(ftp)(http) |
TXT |
| GSE182518_merge_tRNAs_smallRNAseq.txt.gz |
7.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
Less...
More...