Exon junction complex proteins bind nascent transcripts independently of pre-mRNA splicing in Drosophila melanogaster

  1. Subhendu Roy Choudhury
  2. Anand K Singh
  3. Tina McLeod
  4. Marco Blanchette
  5. Boyun Jang
  6. Paul Badenhorst
  7. Aditi Kanhere
  8. Saverio Brogna  Is a corresponding author
  1. University of Birmingham, United Kingdom
  2. Stowers Institute for Medical Research, United States
8 figures and 1 additional file

Figures

Figure 1 with 5 supplements
The EJC core proteins associate with nascent transcripts at polytene chromosomes.

(A) Immunolocalization of EJC proteins (red), eIF4AIII (I-III), Y14 (IV-VI) and MAGO (VII-IX), on salivary gland polytene chromosomes of wandering third instar larvae. Chromosomes were counter-stained with DAPI (blue). Intensity profiles of EJC protein and DAPI signals (III, VI, IX) over a segment at the tip of chromosome 3L (box in merged images) show accumulation of these proteins at interband regions. Scale bar represents 20 µm length. (B) Parallel immunolocalization of EJC proteins (green) and Hrb87F (hnRNP A1) (red) on polytene chromosomes spread from salivary gland without treatment (Control) or after incubation with RNase (RNase treated). Scale bar represent 20 µm length.

https://doi.org/10.7554/eLife.19881.003
Figure 1—figure supplement 1
Characterization of EJC antibodies.

(A) Western blot of S2 cell protein extracts treated with antibodies against eIF4AIII (lane I), MAGO (lane II) and Y14 (lane III) proteins of 46, 17 and 19 kDa expected molecular weight, respectively. Asterisks in lanes 1 and 3 indicate bands of unexpected size, but possibly specific as their intensity is also reduced by RNAi of the target transcript, not shown. (B) Western blot showing that EJC antibodies, used in present study, specifically detect down-regulation of their antigen protein in its RNAi samples. (C) Western blot showing levels of Y14, MAGO and eIF4AIII in nuclear and cytoplasmic fractions of S2 cells. RNA Pol II is shown at the bottom (detected using 8WG16 antibody) as fractionation control; the two expected bands Pol IIo and Pol IIa are indicated. Asterisk indicates a nonspecific band detected in the cytoplasmic fraction. (D) Immunolocalization of eIF4AIII (I-III), Y14 (IV-VI) and MAGO (VII-IX) in whole mount salivary glands of wild-type third instar larvae. Chromosomes were counterstained with DAPI (blue). Scale bar represents 50 µm length.

https://doi.org/10.7554/eLife.19881.004
Figure 1—figure supplement 2
EJC protein signals co-localize with active Pol II.

(A) Magnified view of a chromosome segment of a chromosome showing eIF4AIII (green, I) and Pol II Ser2 (red, II). The split image (III) shows eIF4AIII (green) above and Pol II Ser2 (red) below on same arm. Right panel shows a line drawn on the same segment (IV) and corresponding intensity profile of both of the proteins (V). (B) Double immunostaining of RNA Pol II Ser2 (red) and EJC proteins (green); eIF4AIII (top row), Y14 (middle row) and MAGO (bottom row). Yellow arrows in the middle and bottom row indicate absence of Y14 and MAGO, respectively, at transcription sites marked with intense Pol II Ser2 signal (red). Scale bar represents 20 µm length.

https://doi.org/10.7554/eLife.19881.005
Figure 1—figure supplement 3
Characterization of transgenic flies expressing tagged Y14 or eIF4AIII.

(A) Western blotting of protein extracts of salivary glands from transgenic flies expressing double tagged eIF4AIII-(HA-FLAG)2 detected either with anti-FLAG (lanes, 1 and 2) or with anti-HA antibody (3, 4). (B) Western blotting of protein extracts of salivary glands from transgenes expressing Y14-(HA-FLAG)2 by using anti-FLAG (1, 2) and anti-HA antibodies (3, 4). (C) Immunolocalization of tagged Y14 (red, I, III) and eIF4AIII (red, IV, VI) by using anti-HA antibody, in genotypes indicated on the left of each row. Chromosomes were counterstained with DAPI (blue). Scale bar represents 20 µm length.

https://doi.org/10.7554/eLife.19881.006
Figure 1—figure supplement 4
Y14 and MAGO strictly colocalize at transcription sites.

(A) Parallel immunostaining of tagged Y14 (red) detected with anti-FLAG and endogenous MAGO (green) on polytene chromosomes squash of fkhGAL4>Y14 (HA-FLAG)2. (B) Similar chromosomes immunostaining as in A using anti HA instead of anti-FLAG. Scale bar represents 20 µm length and applies for both panels.

https://doi.org/10.7554/eLife.19881.007
Figure 1—figure supplement 5
Y14 banding pattern differs from that of eIF4AIII.

Parallel immunostaining of tagged eIF4AIII (red) and endogenous Y14 (green) on polytene chromosomes squash of fkhGAL4>eIF4AIII (HA-FLAG)2 larvae. Arrows are indicating sites with strong eIF4AIII but with weak or absent Y14 signal. Chromosomes were counterstained with DAPI (blue).

https://doi.org/10.7554/eLife.19881.008
EJC proteins accumulate at heat shock transcription puffs.

Parallel immunolocalization of hyperphosphorylated RNA Pol II Ser2 (red, II, III, V, VI, VIII, IX) and EJC proteins (green) eIF4AIII (I, III), Y14 (IV, VI), and MAGO (VII, IX), on polytene chromosomes following heat shock at 37°C for 1 hr. Yellow arrowheads indicate accumulation of EJC proteins at the induced intron-containing heat-shock genes, while white arrowheads indicate their accumulation at intronless heat-shock genes. Genes are identified by their map position (details in Results), which are labeled in the merged images on the right panels. Corresponding chromosome arms are mentioned in left column of panel. Scale bar represents 20 µm length.

https://doi.org/10.7554/eLife.19881.009
Figure 3 with 1 supplement
EJC proteins associate with nascent transcripts of both intron and intronless genes.

(A) Immunolocalization of GFP-LacI (red band in boxed areas) at transgene insertion site: S118 (intron plus) on the X (panel I) and of S136 (intron minus) on the 3L (III) chromosome arm. Bands were mapped at 3B for S118 (II) and 63B for S136 (IV) using a standard polytene chromosome map shown above. (B) DAPI-stained (gray) segments of the X chromosome encompassing 3B (I, II) and 3L chromosome encompassing 63B (III, IV), without (I, III) or with (II, IV) ecdysone treatment, which produces a distinct puff at the transgene insertion locus. (C) Immunolocalization of RNA Pol II Ser2 (red) and eIF4AIII (green) at 3B (I-IV and IX-XII) and 63B (V-VIII and XIII-XVI) loci in S118 (I-VIII) and S136 (IX-XVI) transgene following ecdysone treatment. As there is no insert at locus 63B in S118 (V-VIII) and at 3B in S136 (IX-XII), these are used as ecdysone-unresponsive control loci for the transgene at 63B in S136 (XIII-XVI) and for 3B in S118 (I-IV), respectively. Scale bar represents 20 µm length.

https://doi.org/10.7554/eLife.19881.010
Figure 3—figure supplement 1
Y14 associates with nascent transcript of both intron and intronless gene reporters.

Immunolocalization of RNA Pol II Ser2 (red) and Y14 (green) at an intro-containing gene reporter (S118 construct at 3B) (I-IV and IX-XII) and intronless counterpart (S136 at 63B) (V-VIII and XIII-XVI). Both genes were induced by ecdysone treatment as described in Material and methods. As there is no insert at locus 63B in S118 (V-VIII) and at 3B in S136 (IX-XII), these are used as ecdysone-unresponsive control loci for the transgene at 63B in S136 (XIII-XVI) and for 3B in S118 (I-IV), respectively.

https://doi.org/10.7554/eLife.19881.011
Figure 4 with 2 supplements
Y14 associates with expressed genes independent of introns in S2 cells.

(A) Genome-wide average Y14 ChIP-seq enrichment 3000 bases around transcription start sites (TSS) of ChIP (blue trace) versus input DNA (green). (B) Y14 ChIP-seq enrichment (after background subtraction) 2000 bases around transcription start sites (TSS) of expressed (blue trace) or unexpressed (green trace) genes in S2 cells (see Material and methods). (C) Average Y14 enrichment of expressed genes with a different number of introns: 0 to >=5, indicated by traces of different colour (see legend on right of the plot). (D) Average Y14 enrichment across genes as in C but including both expressed and unexpressed genes. (E) Representative chromosome region showing Y14 ChIP-seq enrichment profile (blue), versus that of input DNA (grey). Genes are labeled following Flybase nomenclature.

https://doi.org/10.7554/eLife.19881.012
Figure 4—figure supplement 1
Y14 association with transcribed genes does not correlate with mRNA levels.

Average Y14 enrichment of genes either not expressed (cyan colored line; RPKM = 0) or expressed at different levels (RPKM ranging from 1 to more than 100; indicated by traces of different color).

https://doi.org/10.7554/eLife.19881.013
Figure 4—figure supplement 2
EJC proteins do not co-purify with RNA Pol II.

Immunoprecipitation (IP) with RNA Pol II Ser2 antibody following either RNase or mock treatment. Proteins were assayed by Western blotting for EJC proteins eIF4AIII, Y14 and MAGO, along with elongation factor Spt6 and Hrb87F (hnRNPA1). Goat IgG was used in a parallel IP as background control.

https://doi.org/10.7554/eLife.19881.014
EJC proteins associate with nascent transcripts independently of CWC22 (NCM).

(A) Real-time PCR quantification of NCM RNA level in salivary glands of tubGAL80ts; +; FkhGAL4>NCM-RNAi (right) relative to that in wild type (left). (B) Thirst instar larva and their salivary glands of different genotypes mentioned above each lane. The line indicates a fragment of fat body (fb) adhering to the glands. (C) Immunolocalization of EJC proteins eIF4AIII and Y14 in whole salivary gland cells (I-IV, IX-XII) and at polytene chromosomes (V-VIII, XIII-XVI). The two panels on the left are from wild-type larvae and the two on the right are from tubGAL80ts; +; fkhGAL4/NCM-RNAi larvae. In the mutant (III, IV, XI, XII) white arrows indicate relatively large-sized nuclei while yellow arrows indicate small-sized nuclei. The white line (panel III) indicates a fat body (fb) cell nucleus attached with salivary gland. Chromosomes were counterstained with DAPI (blue). Scale bars represent 20 µm length for corresponding set of images.

https://doi.org/10.7554/eLife.19881.015
Figure 6 with 1 supplement
eIF4AIII is not required for the association of Y14 and MAGO with nascent transcript.

(A) Immunolocalization of EJC proteins (red) eIF4AIII and Y14 in whole salivary gland (I-IV, IX-XII) and polytene chromosomes (V-VIII, XIII-XVI), in wild type (left panels) and tubGAL80ts; +; fkhGAL4/eIF4AIII-RNAi (right panels) larvae. Insets (XV, XVI) are showing a magnified view of the areas enclosed by the white boxes. Chromosomes were counterstained with DAPI (blue). Scale bar represents 20 µm length. (B) Real-time PCR quantification of eIF4AIII and Y14 mRNA levels in fkhGAL4>eIF4AIIIRNAi relative to wild-type larval salivary glands. (C) Western blotting showing eIF4AIII protein levels in tubGAL80ts; +; fkhGAL4/eIF4AIII-RNAi and wild-type glands. (D) Western blotting showing Y14 protein level in fkhGAL4>Y14RNAi and wild-type salivary glands. (E) Western blotting showing level of MAGO protein in fkhGAL4>MAGO-RNAi and wild-type salivary glands. Tubulin was detected as loading control. (F) Immunolocalization of Y14 (red, I, II, IX, X), MAGO (red, III, IV, VII, VIII) and eIF4AIII (red, V, VI, XI, XII) on polytene chromosomes from fkhGAL4>Y14RNAi (left panels) and fkhGAL4>MAGO-RNAi (right panels) larvae. Chromosomes were counterstained with DAPI (blue). Yellow arrows (II, X) indicate accumulation of corresponding proteins at the nucleolus. Scale bar represents 20 µm length.

https://doi.org/10.7554/eLife.19881.016
Figure 6—figure supplement 1
Depletion of eIF4AIII does not affect the pattern of recruitment of Y14 at Pol II transcription sites.

Coimmunolocalization of Y14 (green) and RNA Pol II Ser2 (red) on salivary gland polytene chromosomes of tubGAL80ts; +; fkhGAL4/eIF4AIII-RNAi larvae. Line profile (V) is showing the intensity of both signals across a line drawn on a defined chromosome segment (indicated by the white box, IV), along the DAPI signal (in blue).

https://doi.org/10.7554/eLife.19881.017
eIF4AIII but not Y14-MAGO associates with ribosome-loaded mRNA in S2 cells.

(A) Polysome profiling of cytoplasmic extracts of S2 cells, and Western blotting of corresponding fractions, show distribution of eIF4AIII (upper lane), Y14 (middle lane) and MAGO (lower lane). (B) Polysome profiling and Western blotting as shown in A, following RNase treatment. (C) Polysome profiling and Western blotting following EDTA treatment. (D) Polysome profiling and Western blotting following puromycin treatment at either 4°C (black line) or at room temperature (red line). The red arrows in C and D point to a faster migrating double band of eIF4AIII.

https://doi.org/10.7554/eLife.19881.018
Y14 and MAGO are required for the stability of each other.

(A) Western blotting of total cell lysate from S2 cells to detect eIF4AIII, Y14, MAGO, following eIF4AIII-RNAi, MAGO-RNAi, Y14-RNAi and in untreated cultures. Tubulin was detected as loading control. (B) Real-time PCR quantification of the mRNA level of indicated transcripts was carried out in MAGO-RNAi (left), Y14-RNAi (middle) and eIF4AIII-RNAi (right) along with untreated S2 cells.

https://doi.org/10.7554/eLife.19881.019

Additional files

Supplementary file 1

Table of PCR primers used in the study.

Sequence of primers for real-time RT-PCR or amplification of dsDNA fragment for in-vitro transcription of dsRNAs used for RNAi. Labels refer to gene names.

https://doi.org/10.7554/eLife.19881.020

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  1. Subhendu Roy Choudhury
  2. Anand K Singh
  3. Tina McLeod
  4. Marco Blanchette
  5. Boyun Jang
  6. Paul Badenhorst
  7. Aditi Kanhere
  8. Saverio Brogna
(2016)
Exon junction complex proteins bind nascent transcripts independently of pre-mRNA splicing in Drosophila melanogaster
eLife 5:e19881.
https://doi.org/10.7554/eLife.19881