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. 2015 Sep;35(18):3104-15.
doi: 10.1128/MCB.00473-15. Epub 2015 Jun 29.

Concentration and Localization of Coexpressed ELAV/Hu Proteins Control Specificity of mRNA Processing

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Free PMC article

Concentration and Localization of Coexpressed ELAV/Hu Proteins Control Specificity of mRNA Processing

Emanuela Zaharieva et al. Mol Cell Biol. .
Free PMC article

Abstract

Neuronally coexpressed ELAV/Hu proteins comprise a family of highly related RNA binding proteins which bind to very similar cognate sequences. How this redundancy is linked to in vivo function and how gene-specific regulation is achieved have not been clear. Analysis of mutants in Drosophila ELAV/Hu family proteins ELAV, FNE, and RBP9 and of genetic interactions among them indicates that they have mostly independent roles in neuronal development and function but have converging roles in the regulation of synaptic plasticity. Conversely, ELAV, FNE, RBP9, and human HuR bind ELAV target RNA in vitro with similar affinities. Likewise, all can regulate alternative splicing of ELAV target genes in nonneuronal wing disc cells and substitute for ELAV in eye development upon artificially increased expression; they can also substantially restore ELAV's biological functions when expressed under the control of the elav gene. Furthermore, ELAV-related Sex-lethal can regulate ELAV targets, and ELAV/Hu proteins can interfere with sexual differentiation. An ancient relationship to Sex-lethal is revealed by gonadal expression of RBP9, providing a maternal fail-safe for dosage compensation. Our results indicate that highly related ELAV/Hu RNA binding proteins select targets for mRNA processing through alteration of their expression levels and subcellular localization but only minimally by altered RNA binding specificity.

Figures

FIG 1
FIG 1
Mutants of Drosophila ELAV family RBPs display distinct phenotypes but converge in the regulation of synaptic growth. (A to D) Axonal projections in control, elav (elave5/Y), elav fne (elave5 fneΔ/Y), and elav fne Rbp9 (elave5 fneΔ/Y; Rbp9P2690) embryos were stained with MAb BP102. Arrowheads indicated projection defects and/or irregular positioning of neuromeres. Scale bar, 25 μm. (E to L) Neuromuscular junctions at muscle 13 of control, elav (elave5/elavts1), fne (fneΔ/Df (1)ED7165), Rbp9 (Rbp9P2690/Df(2L)ED206), elav fne (elave5 fneΔ//elavts1 fneΔ), elav; RBP9 (elave5/elavts1; Rbp9P2690/Df(2L)ED206) and fne; Rbp9 (fneΔ/Df (1)ED7165; Rbp9P2690/Df(2L)ED206) third-instar larvae were stained with anti-HRP, and type 1b boutons were quantified (n = 15 to 30) (L). Scale bar, 25 μm. elave5/elavts1 mutants were raised at the permissive temperature during embryogenesis. Statistically significant differences compared to the control values are indicated by asterisks (**, P < 0.01; ***, P < 0.001). (M to Q) Mushroom bodies of control, elav (elave5/elavts1) (flies raised at the permissive temperature during embryogenesis), fne (fneΔ/Df (1)ED7165), Rbp9 (Rbp9P2690/Df(2L)ED206), and fne; Rbp9 (fneΔ/Df (1)ED7165; Rbp9P2690/Df(2L)ED206) adult flies were stained with anti-Fas2. Arrowheads indicate fused beta lobes. Scale bar, 25 μm. (R to V) Photoreceptors of control, elav (elave5; whole-eye clone), fne (fneΔ/Df (1)ED7165), Rbp9 (Rbp9P2690/Df(2L)ED206), and fne RBP9 (fneΔ/Df (1)ED7165; Rbp9P2690/Df(2L)ED206) adult flies from paraffin sections were visualized by autofluorescence. Scale bar, 5 μm. (W to AB) Horizontal paraffin sections of adult heads from control, fne (fneΔ/Df (1)ED7165), Rbp9 (Rbp9P2690/Df(2L)ED206), and fne; Rbp9 (fneΔ/Df (1)ED7165; Rbp9P2690/Df(2L)ED206) 40-day-old adult flies and from elav (elave5/elavts1) (flies raised at the permissive temperature during embryogenesis) 1-day-old (AA) and 7-day-old (AB) adult flies were visualized by autofluorescence. Arrowheads indicate vacuolization, and the asterisks indicate the nonrotated medulla. Scale bar, W 50 μm. (AC) Longevity of control, elav (elave5/elavts1) (flies raised at the permissive temperature during embryogenesis), fne (fneΔ/Df (1)ED7165), Rbp9 (Rbp9P2690/Df(2L)ED206), and fne; Rbp9 (fneΔ/Df (1)ED7165; Rbp9P2690/Df(2L)ED206) flies is shown as the mean from three replicates (20 flies each) with the standard error. (AD) Negative geotaxis of 1-day-, 10-day-, and 20-day-old elav (elave5/elavts1) (flies raised at the permissive temperature during embryogenesis), fne (fneΔ/Df (1)ED7165), Rbp9 (Rbp9P2690/Df(2L)ED206), and fne; Rbp9 (fneΔ/Df (1)ED7165; Rbp9P2690/Df(2L)ED206) adult flies is shown as the mean from three experiments with the standard error normalized to the performance of control flies (set to 100%). Statistically significant differences of comparisons to control fly values are indicated (***, P < 0.001).
FIG 2
FIG 2
Loss of FNE and RBP9 does not affect alternative splicing of ELAV target genes erect wing, neuroglian, and armadillo. (A) Analysis of neuronal alternative splicing in the ewg, nrg, and arm genes in fne; Rbp9 double mutants by RT-PCR. n, neuronal isoform; c, canonical isoform. (B) Schematic of the ELAV-responsive nrg GFP reporter UNGA. (C to E) Alternative splicing of nrg from the UNGA reporter, visualized by anti-GFP staining, is not affected in photoreceptor neurons of fne; Rbp9 mutants but is dramatically reduced in elavedr mutants. Scale bar, 50 μm. (F to I) Alternative splicing of nrg from the UNGA reporter is not affected in neurons of the third-instar larval or adult brain in fne; Rbp9 mutants, which were visualized with anti-GFP staining (top row) and in comparison to anti-ELAV staining (middle and bottom rows). Note the complete overlap between ELAV expression and GFP from the spliced UNGA reporter in fne; Rbp9 mutants (bottom rows of panels F to I). Scale bar, 100 μm.
FIG 3
FIG 3
Binding of recombinant ELAV/Hu family RBPs to RNA of the ELAV target ewg. (A) SDS-polyacrylamide gel showing Coomassie blue-stained recombinant ELAV family RBPs used for binding assays. For each of the recombinant proteins, 0.5 μg, 1 μg, and 2 μg were loaded. Marker proteins were bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (30 kDa). A bacterial protein copurifying with rFNE (lanes 7 to 9) is indicated by the star next to lane 9. (B) EMSA gel with RNA from the ELAV binding site in ewg (pA2-I). Uniformly 32P-labeled RNAs (100 pM) were incubated with recombinant proteins (2 nM, 8 nM, 32 nM, 125 nM, and 500 nM) and separated on 4% native polyacrylamide gels. (C) Graphic representation of EMSA data. The percentage of bound RNA [(input RNA − unbound RNA)/input RNA × 100] is plotted against the concentration of recombinant proteins (in molar units) presented as log values.
FIG 4
FIG 4
Elevated levels of FNE, RBP9, and HuR can regulate alternative splicing of ELAV targets. (A) Expression of HA-tagged ELAV (e.g., HAELAV), FNE, RBP9, and HuR from UAS-containing transgenes in adults with nsyb-GAL4 by Western blotting detection with anti-ELAV antibodies. (B) Neuronal alternative splicing of ELAV targets ewg intron 6 from exon H to J, nrg and arm induced by expression of HA-tagged ELAV, FNE, RBP9, and HuR from UAS-containing transgenes in wing discs with dpp-GAL4 as assessed by RT-PCR. c, canonical; n, neuronal. (C to I) Neuronal alternative splicing of the nrg GFP reporter UNGA upon expression of HA-tagged ELAV, FNE, RBP9, and HuR from UAS-containing transgenes in wing discs with dpp-GAL4. Staining with anti-GFP and anti-HA is as indicated on the left. Due to temporally regulated expression of dpp-GAL4 and because expression of ELAV proteins precedes GFP expression, signals of ELAV proteins and GFP do not entirely overlap. Note that the distantly related poly(U) binding protein Hfp (H) and the SR protein B52 (I) do not induce UNGA splicing. Scale bar, 150 μm. (J to M) Cellular localization of HA-tagged ELAV, FNE, RBP9, and HuR from UAS-containing transgenes in wing discs with dpp-GAL4. Staining with anti-HA and DAPI is as indicated on the left. Scale bar, 10 μm. (N to S) Neuronal alternative splicing of the nrg GFP reporter UNGA upon expression of HA-tagged ELAV, FNE, RBP9, and HuR from UAS-containing transgenes in wing discs with dpp-GAL4 in the presence of temperature-sensitive inhibitor of GAL4, GAL80ts, expressed from a UAS transgene at 18°C, 25°C, and 29°C. Staining in panels N to P with anti-GFP and anti-HA is as indicated on the left. Merged images are shown in the bottom row of panels N to P and in panels Q to S. Scale bar, 150 μm. (T) Quantification of UNGA-splicing shown as means with the standard error from five wing discs.
FIG 5
FIG 5
Rescue of eye development by ELAV/Hu family RBPs in elav mutant eyes. (A) Schematic of the eFVGU elav rescue construct. FRT-mediated recombination results in loss of elav and GAL4 expression under the elav promoter. (B to D) Eye and eye discs of elave5 eFVGU; eyflp males. Neurons shown in panels C and D were stained with anti-ELAV and MAb 24B10, respectively. (E to J) Eyes of wild-type and elave5 eFVGU; eyflp males expressing ELAV/Hu family RBPs or Sxl from UAS transgenes. (K) Quantification of the eye size shown in panels B and E to J. Statistically significant rescue compared to results in the absence of a UAS transgene is indicated (***, P < 0.001).
FIG 6
FIG 6
FNE, RBP9, and HuR can replace neuronal ELAV function under the control of the elav gene. (A) Schematic of the elav rescue construct elav-HA-ELAV. (B) Expression of HA-tagged ELAV (e.g., HAELAV), FNE, RBP9, and HuR under the control of the elav gene in adult flies was determined by Western blotting detection with anti-HA antibodies. In lane 2, HA-tagged ELAV has a larger size due to the presence of the HA tag. (C) Rescue of adult viability of strong hypomorph elavts1 by expression of HA-tagged ELAV, FNE, RBP9, HuR, and NLSRBP9 under the control of the elav gene (n = 200 to 400). (D) Rescue of synaptic growth in elave5/elavts1 flies (raised at the permissive temperature during embryonic development) by expression of HA-tagged ELAV, FNE, RBP9, and HuR under the control of the elav gene is shown as the mean plus standard error of the mean of the number of type 1b boutons at muscle 13 (n = 15 to 28). (E to S) Cellular localization of HA-tagged ELAV, FNE, RBP9, HuR, and NLSRBP9 under the control of the elav gene in larval ventral nerve cord midline neurons. Staining with anti-HA, DAPI, and anti-ELAV is as indicated at the top of the panels. Arrowheads point toward neurons, where NLSHARBP9 is predominantly nuclear, while ELAV becomes cytoplasmic. Scale bar, 10 μm.
FIG 7
FIG 7
Sxl can induce alternative splicing of ELAV target nrg, and ELAV family RBPs can interfere with sexual differentiation and dosage compensation. (A to C) Neuronal alternative splicing of the nrg GFP reporter UNGA in control wing discs and upon expression of UAS-HA-ELAV or UAS-Sxl with dpp-GAL4 stained with anti-GFP antibodies. Scale bar, 150 μm. (D to I) Expression of ELAV with dsx-GAL4 inhibits sexual differentiation of male genitals (side and back views in panels D and E panels F and G, respectively) and sex combs (H and I). Scale bars, 100 μm (E and G) and 50 μm (I). (J) Viability of males from neuronal overexpression of UAS transgenes with elav-GAL4C155, shown as percentage relative to females from the same cross. The total number of flies is shown in parentheses. (K) Viability of females from crosses of mutants in ELAV family proteins with Sxl7B0 null males, shown as a percentage relative to balancer-carrying females (elav) or to males (fne and Rbp9) from the same cross. The total number of flies is shown in parentheses.
FIG 8
FIG 8
Model for target selectivity and functional diversification of ELAV/Hu family RBPs. Circles represent the complement of targets for ELAV, FNE, and RBP9, and overlapping areas indicate shared targets. Main determinants of target selectivity are concentration, binding activity, and subcellular localization.

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