The Drosophila Nab2 RNA binding protein inhibits m6A methylation and male-specific splicing of Sex lethal transcript in female neuronal tissue
- Department of Biology, Emory University, United States
- Department of Cell Biology Emory University School of Medicine, United States
- Graduate Program in Genetics and Molecular Biology, Emory University, United States
- Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University, United States
- Emory Institutional Research and Academic Career Development Award (IRACDA), Fellowships in Research and Science Teaching (FIRST) Postdoctoral Fellowship, United States
- Department of Biochemistry, Duke University School of Medicine, United States
- Department of Neurobiology, Duke University School of Medicine, United States
Figures
Figure 1 with 1 supplement
RNA sequencing detects effects of Nab2 loss on the head transcriptome.
(A) Principal component analysis (PCA) of RNA-seq data from three biological replicates of control and Nab2 mutant (Nab2ex3) male and female heads. (B) Correlation scatter plot of log2 fold change …
Figure 1—figure supplement 1
RNA sequencing reads across the Nab2 locus.
Integrative Genomics Viewer (IGV) image of RNA sequencing reads across the Nab2 locus in Nab2ex3 (top tracks) and control (Nab2pex41) adult female and male heads. Intron-exon structure is indicated …
Figure 2 with 1 supplement
Significantly up-/downregulated RNAs in Nab2ex3 heads are enriched for predicated splicing factors.
(A) Gene Set Enrichment Analysis (GSEA) detects enrichment for the ‘RNA splicing’ Gene Ontology (GO) term in up- and downregulated gene sets in both female (top) and male (bottom) Nab2ex3 datasets. …
Figure 2—figure supplement 1
Gene Ontology (GO) term enrichment among Nab2-regulated alternative splicing events.
Chart illustrating GO terms enriched in the ‘molecular function,’ ‘biological process,’ and ‘cellular component’ categories among altered alternative splicing events detected by mixture of isoforms …
Figure 3 with 2 supplements
Sxl alternative splicing and protein levels are disrupted in Nab2ex3 female heads.
(A) Top panel: normal Sxl alternative splicing patterns across exon 2-4 and exon 8-10 regions in female (F) and male (M). Bottom panel: corresponding sequencing reads across the Sxl locus in the …
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Figure 3—source data 1
Original agarose gel showing RT-PCR analysis of Sxl mRNA in the relevant genotypes in Figure 3B.
Shown as original (bottom) and inverted (top) grayscale.
- https://cdn.elifesciences.org/articles/64904/elife-64904-fig3-data1-v1.zip
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Figure 3—source data 2
Original western blot of head lysates labeled in Figure 3D using anti-Sxl antibody.
Boxed areas correspond to regions shown in the main figure.
- https://cdn.elifesciences.org/articles/64904/elife-64904-fig3-data2-v1.zip
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Figure 3—source data 3
Original western blot of head lysates labeled in Figure 3D using anti-Lamin antibody.
Boxed areas correspond to regions shown in the main figure.
- https://cdn.elifesciences.org/articles/64904/elife-64904-fig3-data3-v1.zip
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Figure 3—source data 4
Raw agarose gel showing RT-PCR analysis of Sxl mRNA in the relevant genotypes and tissues shown in Figure 3F.
- https://cdn.elifesciences.org/articles/64904/elife-64904-fig3-data4-v1.zip
Figure 3—figure supplement 1
RNA sequencing reads across the CG13124 and Ih channel loci.
Integrative Genomics Viewer (IGV) images of RNA sequencing reads across CG13124 and Ih channel in Nab2ex3 (top tracks) and control (Nab2pex41) adult female and male heads. Intron-exon structure is …
Figure 3—figure supplement 2
RNA sequencing reads across the tra and dsx loci.
Integrative Genomics Viewer (IGV) images of RNA sequencing reads across tra, dsx, msl-2, and mle in Nab2ex3 (top tracks) and control (Nab2pex41) adult female and male heads. Intron-exon structure is …
Figure 4 with 1 supplement
Alleles of SxlM8 or the Dosage Compensation Complex (DCC) component male-specific lethal-2 (msl-2) rescues Nab2 phenotypes.
(A) A single copy of the SxlM8 allele, which harbors a 110 bp deletion that causes constitutive exon 2-4 splicing, partially suppresses lethality of Nab2ex3, both zygotically and maternally …
Figure 4—figure supplement 1
Additional genetic interactions between Nab2ex3 msl-2, roX1, and mle.
(A) Adult viability of control females (black bar) with one copy of Nab2ex3 in a msl-2 deficient background (msl-2227 truncating allele over Exel7016 deletion), females lacking Nab2 (Nab2ex3, dark …
Figure 5 with 1 supplement
Reduction of the Mettl3 m6A transferase suppresses viability and behavioral defects in Nab2 mutant females.
(A) Percent of control, Nab2ex3, and Nab2ex3,Mettl3null/+ flies that eclose as viable adults (calculated as #observed/#expected). (B) Negative geotaxis of age-matched adult females of the indicated …
Figure 5—figure supplement 1
Genomic PCR confirms the Nab2ex3,Mettl3null recombinant.
Genomic PCR of two different isolates (#10, #26) of the Mettl3null,Nab2ex3 double mutant chromosome balanced over TM6B. (A) and (C) show PCR products generated from each isolate using primers that …
Figure 6 with 4 supplements
Nab2 associates with the Sxl mRNA and inhibits its m6A methylation.
(A) Diagram of exons (E2, E3, E4) and introns (I2 and I3) of the Sxl pre-mRNA annotated to show coding sequence (CDS; black), the retained intronic region in Nab2ex3 female (gray), and location of …
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Figure 6—source data 1
Raw agarose gel showing RT-PCR analysis of Sxl mRNA in the relevant genotypes shown in Figure 6H.
- https://cdn.elifesciences.org/articles/64904/elife-64904-fig6-data1-v1.zip
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Figure 6—source data 2
Raw agarose gel showing RT-PCR analysis of RpL32 mRNA in the relevant genotypes shown in Figure 6H.
- https://cdn.elifesciences.org/articles/64904/elife-64904-fig6-data2-v1.zip
Figure 6—figure supplement 1
Detailed schematic of the exon 2-3-4 Sxl locus with annotated locations of introns and exons annotated to show coding sequence (CDS; blue), the retained intronic region in Nab2ex3 females (gray), and locations of color-coded primer pairs (E2-F and E2-R, E2-F and E4-R, I2-F, and E3-R, I3-F, and E4-R), poly(U) sites red lines, poly(A) sites green lines, and mapped m6A locations in Drosophila embryos yellow lines (Kan et al., 2017).
Colored dotted lines indicated sex-specific splicing in wildtype adults and the altered splicing documented in this study. Boxed areas below summarize exon-intron structure in wild type heads and Nab…
Figure 6—figure supplement 2
Nab2 limits m6A methylation of additional RNAs.
Quantitative real-time PCR analysis of Act5c and Usp16 mRNAs present in anti-m6A precipitates of control (Nab2pex41; black), Nab2ex3 (gray), and Mettl3null (dark gray) adult female heads. 1-day-old …
Figure 6—figure supplement 3
Heterozygosity for Mettl3 does not alter Sxl splicing in control or Nab2ex3 mutant female heads.
(A) qRT-PCR analysis of RNAs harvested from adult female heads of the indicated genotypes with primers that amplify spliced Sxl exon 2-exon 3 (E2-E3) or exon 3-exon 4 (E3E4-E4). The E3E4 primer …
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Figure 6—figure supplement 3—source data 1
Raw agarose gel showing RT-PCR analysis of Sxl mRNA in the relevant genotypes shown in Figure 6—figure supplement 3.
Boxes correspond to regions included in the figure supplement.
- https://cdn.elifesciences.org/articles/64904/elife-64904-fig6-figsupp3-data1-v1.zip
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Figure 6—figure supplement 3—source data 2
Raw agarose gel showing RT-PCR analysis of RpL32 mRNA in the relevant genotypes shown in Figure 6—figure supplement 3.
Boxes correspond to regions included in the figure supplement.
- https://cdn.elifesciences.org/articles/64904/elife-64904-fig6-figsupp3-data2-v1.zip
Figure 6—figure supplement 4
Neuronal overexpression of Mettl3 RNA using the Gal4/UAS system.
qRT-PCR analysis of Mettl3 RNA levels in heads of control w1118 (lane 1), or Mettl3 overexpressing (elav-Gal4,UAS-Mettl3) adult females in the absence (lane2) or presence (lane 3) of a single Nab2ex3…
Author response image 1
Author response image 2
Tables
Table 1
Alternative exon use (DexSeq) in Nab2ex3 head transcriptomes.
| Females | Males | |
|---|---|---|
| No. of alternatively used exons * | 151 | 114 |
| * |(exon usage fold change)| >~1.75 BH Adj. p<0.05 | ||
| Top mis-spliced transcripts | ||
| Females | foldΔ exon usage | BH adj. p-value |
| Sex lethal (Sxl) | 2.86 | 3.08 × 10–235 |
| CG13124 | 2.45 | 1.09 × 10–81 |
| Ih channel | 2.29 | 3.28 × 1063 |
| Ace | 1.81 | 1.02 × 10–59 |
| Males | foldΔ exon usage | BH adj. p-value |
| Ace | 2.02 | 1.88 × 10–169 |
| Pkc53E | 1.74 | 6.12 × 10–102 |
| plx | 2.12 | 9.03 × 10–67 |
| Pkn | 1.84 | 9.04 × 10–67 |
| Bacc | 2.31 | 1.11 × 10–64 |
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Table 1—source data 1
Files (.bed format, openable as .excel tables) of splicing defects detected by lower threshold analysis than in Table 1.
Abbreviations: A3’SS, alternative 3′ splice site; A5’SS, alternative 5′ splice site; MEE, mutually exclusive exon; RI, retained intron; SE, skipped exon.
- https://cdn.elifesciences.org/articles/64904/elife-64904-table1-data1-v1.zip
Additional files
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Supplementary file 1
RNA abundance in Nab2ex3 vs. control female and male adult heads.
This file contains a list of all annotated RNAs and their corresponding expression level across all samples in female and male adult heads (i.e., raw counts) and their fold-change in Nab2 null heads relative to control in descending rank order of significance (p-value, column H) as determined by DESeq2 analysis.
- https://cdn.elifesciences.org/articles/64904/elife-64904-supp1-v1.xlsx
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Supplementary file 2
Splicing changes in female and male Nab2 null heads relative to control heads.
This file contains a list of RNAs and corresponding changes in splice patterns in Nab2 null vs. control detected by MISO. A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice site; RI, retained intron; SE, skipped exon.
- https://cdn.elifesciences.org/articles/64904/elife-64904-supp2-v1.xlsx
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Supplementary file 3
DEXSeq called differential exon usage in Nab2 null heads.
This file contains a list of RNAs with differential exon usage in Nab2 null female or male heads detected by DEXSeq in descending rank order of significance (p-value, column P).
- https://cdn.elifesciences.org/articles/64904/elife-64904-supp3-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/64904/elife-64904-transrepform1-v1.docx
