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. 2013 Jul 30;110(31):12750-5.
doi: 10.1073/pnas.1310735110. Epub 2013 Jul 12.

RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee

Hongmei Li-Byarlay  1 , Yang Li, Hume Stroud, Suhua Feng, Thomas C Newman, Megan Kaneda, Kirk K Hou, Kim C Worley, Christine G Elsik, Samuel A Wickline, Steven E Jacobsen, Jian Ma, Gene E Robinson
Affiliations

RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee

Hongmei Li-Byarlay et al. Proc Natl Acad Sci U S A. .

Abstract

Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing.

Keywords: epigenetics; gene regulation; gene silencing; insect.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Noninvasive high-throughput method of delivering RNA interference in vivo. Spraying a nebulized mixture of nanoparticles (PFC-NP) and small interfering RNA (siRNA) on bees allows penetration through the insect tracheal respiratory system. (A) Treated: NP (labeled green with Alexa 488) and dnmt3 siRNA (labeled red with Q-670). (B) Control: NP (green) and GFP siRNA (no label). (C) Quantitative analysis of fluorescence reveals significantly higher levels of red signal in treated (n = 5) comparing to control bees (n = 6, P < 0.001, two-tailed t test), demonstrating penetration via the trachea. (D) Schematic of an inner view of trachea, spiracle atrium, and body wall. Diagram adapted from ref. .
Fig. 2.
Fig. 2.
Effects of spraying a nebulized mixture of nanoparticles and small interfering RNA of dnmt3 on dnmt3 expression ± SEM in honey bee abdominal fat body. Results from three biological replicates (bees from unrelated colonies, n = 22 for colony R12; n = 31 for R62, and n = 33 for R54; two-tailed t tests). Analysis of pooled data: mixed-model ANOVA, df = 82, F = 15.52. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 3.
Fig. 3.
Effects of dnmt3 knockdown on differentially spliced genes. (Upper) Gene model in both splicing isoforms with the number of RNAseq reads mapped to each splicing junction. (Lower) Exon coverage from RNAseq. Blue, control; orange, dnmt3 knockdown. Derivation of the AS ratios in the colored squares are in SI Materials and Methods.
Fig. 4.
Fig. 4.
Effects of dnmt3 knockdown on exon skipping and methylation in gene GB46657 (galactokinase-like). (Top and Middle) Same information as in Fig. 3. (Bottom) mCpG sites with vertical lines, together with percent differences between treatments.
See this image and copyright information in PMC

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