The Drosophila Helicase MLE Targets Hairpin Structures in Genomic Transcripts

Abstract

RNA hairpins are a common type of secondary structures that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi) and microRNA (miRNA) pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss) RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE was shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. Here we report that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, we show that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. We use two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway.

Fig 1. MLE is enriched at the plasmid integration site when transcription of the transgene is active.

Left panel, Polytene chromosomes from male larvae expressing a dsRNA targeting Hrb87F (Hrb87F RNAi) under the induction of Actin5C-GAL4 or larvae in which the production of the dsRNA is not activated (ctrl). MLE paints the X chromosome in both samples and is enriched at the integration site of the plasmid only in Hrb87F RNAi larvae. The white arrows indicate the plasmid integration site. In the right panel is a detail of the region marked by the arrows.

Fig 2. MLE localization at the integration site of the plasmid does not require the MSL complex.

(A) Left panel, Polytene chromosomes from male larvae expressing a dsRNA targeting Hrb87F (Hrb87F RNAi) under the induction of Actin5C-GAL4. MSL1, MSL3 and MOF paint the X chromosome but are absent at the integration site of the plasmid indicated by the white arrows. In the right panel is a detail of the region marked by the arrows. (B) Polytene chromosomes from female larvae expressing a dsRNA targeting Hrb87F (Hrb87F RNAi) following induction with Actin5C-GAL4 and larvae in which the production of the dsRNA is not induced (ctrl).

Fig 3. MLE targets sites of dsRNA transcription in a sequence and chromosome location independent manner.

(A) MLE staining of polytene chromosomes from female larvae expressing either a mof dsRNA or an Hrb87F dsRNA construct from a pValium1 insertion of TRiP line collection following induction with Actin5C-GAL4. (B) GAL4 and MLE staining of polytene chromosomes from female larvae expressing dsRNA constructs integrated respectively at Chr2L 30B3 (Jil1 RNAi) and Chr2L 22A5 (Hp1 RNAi) and induced by Actin5C-GAL4.

Fig 4. MLE is not recruited by high levels of expression.

(A) MLE staining of polytene chromosomes from female larvae expressing either the luciferase gene or a dsRNA targeting the luciferase gene following induction with Actin5C-Gal4. MLE is present at the integration site of the plasmid when the dsRNA is transcribed but not when the luciferase gene is expressed. White arrows indicate the integration site of the plasmid. (B) Luciferase assay in larvae carrying ActGal4-induced and non-induced pValium1 and pValium10-mediated luciferase gene inserts. High levels of luciferase are observed after induction. (C) qRT-PCR analysis of luciferase gene transcript levels in larvae carrying ActGal4-induced and non induced pValium1 and pValium10-mediated luciferase gene inserts. Luciferase gene expression was normalized using pka gene and the results are expressed in terms of fold difference relative to the basal transcript levels observed in pValium1-luciferase sample without induction. The results are the average of three independent biological replicates.

Fig 5. MLE targets shRNA at their site of transcription.

MLE staining of polytene chromosomes from female larvae expressing short hairpin RNAs after induction with Actin5C-GAL4.

Fig 6. RNase treatment strongly reduces MLE signal at the integration site of the plasmid.

(A) Left panel, MLE staining of polytene chromosomes from male larvae expressing a dsRNA targeting Hrb87F after induction with Actin5C-GAL4. The incubation of the salivary glands in RNase A (RNase A) perturbs MLE localization at the integration site of the plasmid while in the absence of RNase A (ctrl) the MLE signal is still highly enriched. The white arrows indicate the plasmid integration site. In the right panel is a detail of the region marked by the arrows. (B) Quantitative analysis of fluorescence levels. MLE signal at the integration site of the plasmid, expressed in terms of corrected total band fluorescence (CTBF), is significantly reduced after RNase A treatment (p value <0.001). The analysis was performed on 33 polytene chromosomes treated with RNase A and 11 control chromosomes.

Fig 7. MLE physically interacts with RNA hairpins.

(A) Schematic representation of the hairpin RNA generated by pValium1-Hrb87F RNAi induction. In blue is the stem formed by the inverted repeats, in black is the white intron. The arrow indicates the primer used to obtain the cDNA and the two fragments analyzed by qPCR (P1 and P3) are indicated by the black lines (B) RIP experiment from extracts of female larvae expressing a dsRNA targeting Hrb87F after induction with Actin5C-GAL4. The results are expressed in terms of fold difference between MLE-associated RNA and IgG-associated RNA normalized for the starting material. Three independent biological replicates are presented for P1 and P3 primer pairs.

Fig 8. Rm62 RNA helicase is not enriched at sites of hairpin RNA transcription.

Left panel, Rm62 staining of polytene chromosomes from female larvae expressing a dsRNA targeting Hrb87F after induction with Actin5C-GAL4. The white arrows indicate the plasmid integration site. The right panel shows a detail of the region marked by the arrows.

Fig 9. MLE mutation affects RNAi efficiency.

(A) Wing phenotypes from female flies in which Notch dsRNA expression is induced by C96-GAL4. Class1: wild type; class 2: missing margin bristles and absent notching; class 3: moderate notching; class 4: extensive notching; class 5: missing most of the wing margins; class 6: complete lack of margins and reduced wing blade. The chart on the right side of the figure represents the Notch RNAi wing phenotypes distribution in wild type, mle homozygous mutant and mle heterozygous mutant background. The results are the sum of three to six independent crosses per genotype. (B) Wing phenotypes from female flies in which Egfr dsRNA expression is induced by ms1096-GAL4. Class1: wild type; class 2: all the veins present; class 3: absent anterior cross vein (acv) or presence of a partial longitudinal vein; class 4: absent acv plus one partial longitudinal vein; class 5: absent acv plus two partial longitudinal veins; class 6: acv vein absent plus one longitudinal vein absent plus one partial longitudinal vein; class 7: most of the veins absent. The chart on the right side of the figure represents the Egfr RNAi wing phenotypes distribution respectively in wild type and mle homozygous mutant background. The results are the sum of four to five independent crosses per genotype.