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. 2003 Oct 15;17(20):2526-38.
doi: 10.1101/gad.1106703. Epub 2003 Oct 1.

ELAV Inhibits 3'-end Processing to Promote Neural Splicing of Ewg pre-mRNA

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Free PMC article

ELAV Inhibits 3'-end Processing to Promote Neural Splicing of Ewg pre-mRNA

Matthias Soller et al. Genes Dev. .
Free PMC article

Abstract

The embryonic lethal abnormal visual system (ELAV) is a gene-specific regulator of alternative pre-mRNA processing in neurons of Drosophila. Here we define a functional in vivo binding site for ELAV in neurons through the development of a reporter gene system in transgenic animals in combination with in vitro binding assays. ELAV binds to erect wing (ewg) RNA 3' of a polyadenylation site in the terminal intron 6. At this polyadenylation site, ELAV inhibits 3'-end processing in vitro in a dose-dependent and sequence-specific manner, and ELAV binding is necessary in vivo to promote splicing of ewg intron 6. Further, the AAUAAA poly(A) complex recognition sequence, together with ELAV, is required to regulate neural 3' splice site choice in vivo. In addition, the use of segmentally labeled RNA substrates in UV cross-linking assays suggest that ELAV does not inhibit or redirect binding of cleavage factor dCstF64 at the regulated polyadenylation site on ewg RNA. These data indicate that binding of 3'-end processing factors, together with ELAV, can regulate alternative splicing.

Figures

Figure 1.
Figure 1.
ELAV switches polyadenylation of ewg pre-mRNA to splicing. (A) 3′ ends of ewg transcripts amplified by nested RT-PCR with RNA extracted from eye discs with no (-), reduced (e), or wild-type (+) ELAV levels. (e) elavedr. The top panel shows the amplified ends of ewg transcripts, and the bottom panel shows a common part of ewg transcripts amplified as a standard. Primers used in the two PCR reactions are indicated on the left and depicted in B. Graphic illustrations of PCR products are shown on the left and indicate the different 3′ ends and pA sites as depicted in B. PCR products were separated on 5% denaturing polyacrylamide gels. (B) Schematic of the ewg rescue reporter gene (tcgER). The transcriptional start is indicated by an arrow in the elav promotor. ORFs are shown as boxes with either a HA or VSV tag at the end. Deletion Δ7 is indicated at the bottom [starts 89 nt after the 5′ splice site, extends to the MfeI site (M) in intron 6, and also contains an HA tag]. pA used are indicated by vertical arrows. RT and return 3′ RACE primers are shown for the pA4 site only. (C) Splicing of ewg intron 6 analyzed by semi-quantitative RT-PCR with eye disc RNA from wild-type and Δ7 tcgER in wild-type (+) or elavedr background (e). A fragment from Appl transcripts was amplified as standard. Graphic illustrations of ewg splice products are shown on the right. Uniformly 32P-dCTP-labeled PCR products were separated on 8% polyacrylamide gels. (D) Southern blot probed with either 32P-end-labeled HA or VSV oligonucleotides to visualize ends of transcripts from wild-type and Δ7 tcgER transgenes amplified by 3′ RACE from eye disc RNA in wild-type (+) and elavedr mutant background (e) as indicated in C. Graphic illustrations of PCR products are shown on the right and indicate the different 3′ ends and pA sites. The shorter fragments detected in lanes 3 and 4 on the HA-Southern are due to the deletion Δ7 and 3′-end formation at pA2.
Figure 2.
Figure 2.
ELAV binds 3′ of the pA2 site on ewg premRNA. (A) Schematic of ewg RNAs used for UV crosslinking assays. Δ8 derives from Δ7, but lacks the HA tag. (B) UV cross-linking of uniformly [32P]ATP-labeled Δ8 RNA in neuronal nuclear extract. Proteins were separated on 10% SDS-polyacrylamide gels after crosslinking and RNase digestion (lane 1) or after IP with anti-ELAV antibodies (lane 2). The control IP (lane 3) was done with protein A/G beads alone. Arrowhead indicates ELAV. (C) UV cross-linking of uniformly [32P]ATP-labeled Δ8 RNA in nuclear extract from Kc cells and heads of wild-type flies or flies expressing the N-terminal truncation mutant RBD60 (lanes 1-3), and anti-ELAV Western of those nuclear extracts (lanes 4-6). A 50-kD protein present in addition to ELAV is marked by a star next to lane 3. Arrowheads indicate ELAV. (D) UV cross-linking of uniformly [32P]ATP-labeled ewg Δ8 and 3′ UTR RNA. (E) UV cross-linking of uniformly [32P]ATP-labeled Δ9-Δ11 RNAs and mutated Δ8 RNAs. AU4-6 motif mutations are depicted in F. At the bottom, labeling intensity of the 50-kD signal from mutant RNAs is compared with parental Δ8 RNA (control, lane 4) and shown as PhosphorImager intensity (PI) from three experiments. Arrowheads indicate ELAV. (F) Schematic of ewg RNA pA2-I (pA2 signal to intron I) showing AU4-6 motif locations (ovals). Crossed oval represents mutated AU4-6 motifs to A(CU)2-3. For sequence see Figure 7A. (Py) Pyrimidine tract. (G) EMSA of RNA pA2-I. The indicated uniformly [32P]ATP-labeled RNAs (100 pM) were incubated with recombinant HA-ELAV (12.5 nM, 50 nM, 0.2 μM, 0.8 μM, and 3.2 μM) or HA-ELAV-AGD (3.2 μM, lane 7). The first lane of each RNA (lanes 1,8,14) shows the input RNA without protein. (H) Graphic representation of EMSA data. The percent of bound RNA (input RNA -unbound RNA/input RNA × 100) is plotted against the concentration of recombinant ELAV (in molar) presented as log from three experiments. (Open squares) Wild-type RNA pA2-I; (filled rectangles) pA2-I RNA with mutation m1-3; (open circles) anti-sense RNA pA2-I.
Figure 7.
Figure 7.
(A) Sequence context around ewg pA2 up to the 3′ splice site of exon I. (B) Schematic of CPSF, CstF, and ELAV binding to ewg pre-mRNA. In A, the AAUAAA sequence, tandem AU4-6 motifs, and the AG dinucleotide from the 3′ splice site at exon I are in capital letters. A G/U-rich element is underlined and the polypyrimidine tract is in italics. The cleavage site is indicated with an asterisk. AU4-6 motifs are in bold and tandem AU4-6 motifs are aligned to a putative consensus sequence evident within the ELAV binding sites.
Figure 3.
Figure 3.
ELAV inhibits 3′-end formation at pA2 in vitro. (A) Schematic of ewg 299-nt substrate RNA pA2-ivs used for in vitro cleavage/pA. The m1-3 mutation is as in Figure 2F; ΔAAUAAA deletes those nucleotides and +54 adds 54 nt at the 3′ end. (B) Cleavage/pA assay of ewg wild-type and mutant pA2-ivs RNAs as indicated above the panel in nonneuronal nuclear extract. Time points were 0, 25, and 50 min. The 5′ cleavage product is marked with an arrowhead. (M) RNA size marker. (C) Longer exposure of upper parts of the gel from B shows the addition of poly(A) tails in the absence of 3′dATP and results in RNAs longer than the substrate (lanes 14,15). (D, top panel) Amplification of 5′ cleavage fragments from in vitro cleavage/pA assays in the presence (+) or absence (-) of 3′dATP by oligo dT-mediated RT-PCR (3′ RACE). (Bottom panel) Control RT-PCR to show equal input was done with a RT/return primer in the 3′ part of pA2-ivs substrate RNA to amplify uncleaved substrate RNA. Fragments were separated on 3% agarose gels. (M) DNA size marker. (E) Cleavage/pA assay of ewg wild-type and mutant pA2-ivs RNAs as indicated above the panel in nonneuronal nuclear extract. Concentration of recombinant ELAV is 0.2, 0.4, and 0.8 μM and of ELAV-AGD is 0.8 μM. (F) Cleavage/pA assay of wild-type and mutant substrate pA2-ivs RNAs in neuronal nuclear extract. Time points were 0, 20, 40, and 60 min.
Figure 6.
Figure 6.
dCstF64 binds to ewg pA2 in the presence of ELAV. (A) Western showing expression of dCstF64-HA in eye discs using GMR-GAL4 driver from 0, 1, or 2 UAS transgenes (lanes 1-3, respectively), and UV cross-linking of pA2-ivs RNA in neuronal nuclear extract prepared from wild-type fly heads (-, lane 4) and fly heads expressing dCstF64-HA with elav-GAL4 driver (+, lane 5). (B) UV cross-linking of Δ8 RNA in neuronal nuclear extract containing dCstF64-HA and IP with anti-HA antibodies (lane 3). Control IP was done with protein A/G beads alone. The arrow marks dCstF64-HA and the arrowhead points toward ELAV. (C) Schematic of segmentally 32P-labeled pA2-ivs RNAs used in UV cross-linking assays in D and E. A G/U-rich sequence element as a putative dCstF64-binding site is indicated below pA2-ivs. (D,E) UV cross-linking assays with partially 32P-labeled pA2-ivs RNAs in neuronal nuclear extract containing dCstF64-HA, and IP with anti-ELAV and anti-HA antibodies. Added recombinant ELAV (0.16 μM, 0.32 μM, and 0.64 μM) to UV cross-linking extracts and labeled RNA are indicated above the panel. As a control, UV cross-linking reactions with pA2-ivs C or pA2-ivs were incubated with protein A/G beads alone (D, lanes 7,20; E, lanes 1,12). The arrow marks dCstF64-HA and the arrowhead points toward ELAV. (F) Coimmunopreciptation of ELAV and dCstF64 from neuronal nuclear extract expressing UAS dCstF64-HA with elav-GAL4 driver. (Lanes 1-3) IP with anti-ELAV antibodies and detection of dCstF64-HA (arrow) with anti-HA antibodies. (Lanes 4-6) IP with anti-HA antibodies and detection of ELAV (arrowhead). (Lanes 2,5) Control IP was with protein A/G beads alone. Twenty-five percent input of nuclear extract proteins is shown in lanes 1 and 4.
Figure 4.
Figure 4.
Inhibition of 3′-end formation at pA2 in neurons depends on ELAV binding in vivo. (A) Schematic of ewg intron 6 as present in tcgER reporter gene. (B) Splicing of ewg intron 6 analyzed by RT-PCR with eye disc RNA from wild-type and m1-3 (mutations in AU4-6 motifs) tcgER in wild-type (+) or elavedr mutant background (e). A fragment from Appl transcripts was amplified as standard. Graphic illustrations of ewg splice products are shown on the right. (C) Southern blot probed with either a 32P-end-labeled HA or VSV oligonucleotide to visualize 3′ ends of transcripts from wild-type and m1-3 tcgER amplified by 3′ RACE from eye disc RNA in wild-type (+) or elavedr mutant background (e) as indicated in Figure 4B. Graphic illustrations of PCR products are shown on the right and indicate the different 3′ ends and pA sites.
Figure 5.
Figure 5.
The AAUAAA pA recognition sequence is required for neural 3′ splice site choice. (A) Schematic of ewg intron 6 as present in tcgER reporter gene. (B) Splicing of ewg intron 6 analyzed by RT-PCR with eye disc RNA from wild-type and ΔpA2 (AAUAAA deleted at pA2) tcgER in wild-type (+) or elavedr mutant background (e). A fragment from Appl transcripts was amplified as standard. Graphic illustrations of ewg splice products are shown on the right. (C) Southern blot probed with either a 32P-end-labeled HA or VSV oligonucleotide to visualize 3′ ends of transcripts from wild-type and ΔpA2 tcgER amplified by 3′ RACE from eye disc RNA in wild-type (+) or elavedr mutant background (e) as indicated in B. Graphic illustrations of PCR products are shown on the right and indicate the different 3′ ends and pA sites. (D) RT-PCR from eye disc RNA from wild-type flies containing one, two, or four copies of wild-type tcgER transgenes. A fragment from Appl transcripts was amplified as a standard, and a fragment amplified from the 5′ end of the reporter transcript (eeF/R) shows the amount of transcripts.

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