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. 2005 Sep;25(17):7580-91.
doi: 10.1128/MCB.25.17.7580-7591.2005.

ELAV Multimerizes on Conserved AU4-6 Motifs Important for Ewg Splicing Regulation

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Free PMC article

ELAV Multimerizes on Conserved AU4-6 Motifs Important for Ewg Splicing Regulation

Matthias Soller et al. Mol Cell Biol. .
Free PMC article

Abstract

ELAV is a gene-specific regulator of alternative pre-mRNA processing in Drosophila neurons. Since ELAV/Hu proteins preferentially bind to AU-rich regions that are generally abundant in introns and untranslated regions, it has not been clear how gene specificity is achieved. Here we used a combination of in vitro biochemical experiments together with phylogenetic comparisons and in vivo analysis of Drosophila transgenes to study ELAV binding to the last ewg intron and splicing regulation. In vitro binding studies of ELAV show that ELAV multimerizes on the ewg binding site and forms a defined and saturable complex. Further, sizing of the ELAV-RNA complex and a series of titration experiments indicate that ELAV forms a dodecameric complex on 135 nucleotides in the last ewg intron. Analysis of the substrate RNA requirements for ELAV binding and complex formation indicates that a series of AU(4-6) motifs spread over the entire binding site are important, but not a strictly defined sequence element. The importance of AU(4-6) motifs, but not spacing between them, is further supported by evolutionary conservation in several melanogaster species subgroups. Finally, using transgenes we demonstrate in fly neurons that ELAV-mediated regulation of ewg intron 6 splicing requires several AU(4-6) motifs and that introduction of spacer sequence between conserved AU(4-6) motifs has a minimal effect on splicing. Collectively, our results suggest that ELAV multimerization and binding to multiple AU(4-6) motifs contribute to target RNA recognition and processing in a complex cellular environment.

Figures

FIG. 1.
FIG. 1.
ELAV binds as a multimer to ewg RNA pA2-I. (A) Schematic of ELAV and the N-terminal truncation mutant RBD60. (B) Coomassie blue-stained SDS gel of recombinant ELAV (50 kDa) and RBD60 (40 kDa). Protein was loaded at 1.25, 2.5, 5, and 10 μg. Marker proteins were bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa). (C) Binding of recombinant ELAV and RBD60 to poly(U) RNA captured by DEAE-Sepharose beads and detection by Western blotting. sn, supernatant. (D) Schematic of the last ewg intron 6 and 3′ UTR with splicing and polyadenylation choices. The two major isoforms are those with intron 6 spliced from H to J in the presence of ELAV (solid line) or with polyadenylation in intron 6 at pA2 (boldface) in the absence of ELAV. (E) Magnification of the ELAV binding region used as substrate RNA pA2-I, depicting polyadenylation site pA2 and part of exon I as well as AU4-6 motifs in sequence elements m1, m2, and m3. Py, polypyrimidine tract. (F) EMSA of ewg RNA pA2-I with ELAV and RBD60 protein. Uniformly 32P-labeled RNA pA2-I (100 pM) was incubated with recombinant ELAV or recombinant RBD60 (1.38 nM, 5.5 nM, 22 nM, 87.5 nM, 0.35 μM, and 1.4 μM) and separated on 4% native polyacrylamide gels. Lane 1 shows input RNA without protein. (G) Graphic representation of EMSA data from ELAV and RBD60 binding to RNA pA2-I. The percentage of bound RNA (input RNA − unbound RNA/input RNA × 100) is plotted against the concentration of recombinant ELAV or RBD60. Results are from three experiments. Error bars indicate standard deviations.
FIG. 2.
FIG. 2.
ELAV tetramerizes and forms a 700-kDa complex with ewg RNA pA2-I. (A and B) Superose 6 gel filtration of ELAV or RBD60 complexes. ELAV (A) or RBD60 (B) RNA-pA2-I complexes were formed with 10 fmol 32P-labeled RNA pA-I and 320 pmol ELAV or RBD60 in 100 μl and loaded onto the gel filtration column. The top panels show an exposure of the RNA elution profile and the middle panels a Western blot of the elution profile for ELAV (A) or RBD60 (B). The bottom panels show a quantification of RNA (dashed line) and ELAV (A) or RBD60 (B) elution profiles (solid line) in conjunction with prerun standards. Peaks of free ELAV or RBD60 are numbered according to their multimerization state (1, monomer; 2, dimer; and 4, tetramer). The protein peak is slightly retarded compared to the RNA peak, which might be due to the length of the run and substoichiometric stability of the complex (see Fig. 3A and B). (C) Superose 6 gel filtration of RNA pA2-I, ELAV, or RBD60 alone. The top panel shows an exposure of the RNA elution profile, and the two panels below show Western blots of the elution profile for ELAV or RBD60. The bottom panel shows a quantification of RNA (dotted line) and ELAV (solid line) or RBD60 (dashed line) elution profiles in conjunction with prerun standards. Peaks of free ELAV or RBD60 are numbered according to their multimerization state (1, monomer; 2, dimer; and 4, tetramer). (D) Determination of multimerization states of ELAV and RBD60 by cross-linking. Recombinant ELAV and RBD60 were cross-linked at two concentrations (3 μM and 0.75 μM) with BM[PEO]3 in a maleimide reaction starting at equimolar concentrations over sulfhydryl groups (6, 18, and 36 μM and 1.5, 4.5 and 13.5 μM, respectively). Proteins were separated on a 7% SDS gel and detected by Western blotting with an anti-ELAV antibody. Lanes 1 and 8 show input protein without cross-linker (same preparation as in Fig. 1B), and the multimerization state is labeled at the right of ELAV and RBD60 protein lanes, respectively. Since about 5 to 10% of a shorter proteolysis fragment is present, the lower of the two dimer bands might include this fragment, which is, however, not observed with tri- and tetramer adducts. The positions of the two cysteines in ELAV are depicted in a schematic of ELAV above the gel.
FIG. 3.
FIG. 3.
ELAV binds as a dodecamer to one ewg RNA pA2-I. (A and B) Stoichiometry EMSA of ewg RNA pA2-I with ELAV or RBD60 protein. Recombinant ELAV (A) or RBD60 (B) was incubated with trace 32P-labeled RNA pA2-I (0.2 μM) well above the dissociation constant (17 nM) and separated on 4% native polyacrylamide gels. Protein concentrations are shown above the gel. Lane 1 shows input RNA without protein. The arrow in A points towards a semistable intermediate probably representing a tetramer. The final complex forms at an RNA/protein ratio of between 1:8 and 1:9 (compare lanes 8 and 9 in A and B, and data not shown). (C) Graphic representation of EMSA data from ELAV and RBD60 binding to RNA pA2-I above the dissociation constant as shown in A and B. The percentage of bound RNA (input RNA − unbound RNA/input RNA × 100) is plotted against the molar equivalent of protein to RNA. (D) Titration EMSA of ELAV against RBD60 with RNA pA2-I at complex-forming concentrations. Uniformly 32P-labeled RNA pA2-I (100 pM) was incubated with recombinant ELAV, recombinant RBD60, or a combination of both at a final concentration of 3.2 μM. The recombinant ELAV concentrations in successive lanes are 0%, 5%, 10%, 20%, 40%, 60%, 80%, and 100% (the concentrations of RBD60 are accordingly reduced). The first lane shows the complex with RBD60 alone, and the last lane shows the complex with ELAV alone. Complexes were separated on 4% native polyacrylamide gels. (E) EMSA of ELAV with two separable substrate RNAs. Uniformly 32P-labeled RNA pA2-I (100 pM), two-copy RNA pA2-I (100 nM), or a mixture of both (50 pM each) was incubated with recombinant ELAV at a concentration of 3.2 μM (lanes 4 to 6) and separated on 4% native polyacrylamide gels. Input RNAs are shown in lanes 1 to 3.
FIG. 4.
FIG. 4.
Multiple sequence elements spread over 135 nucleotides contribute to ELAV complex binding on ewg RNA. (A) Schematic of ewg substrate RNA pA2-I (from polyadenylation site pA2 to exon I). AU4-6 motifs are indicated as ovals in sequence elements m1, m2, and m3. Py, polypyrimidine tract. (B) Deletions in RNA pA2-I used for EMSA analysis. RNAs used were from 67 to 213 nt long, including 43 nt of vector sequence (see Materials and Methods for sequence). Averaged binding affinities from at least three EMSA experiments are shown on the right. (C and D) Representative gels of EMSA analysis using various uniformly 32P labeled substrate RNAs (100 pM) as indicated at the top. ELAV concentrations were 12.5 nM, 50 nM, 0.2 μM, 0.8 μM, and 3.2 μM, except 3.2 μM for pA2-I + and Δ134 (lanes 19 and 20 in D). Absence of ELAV is indicated above the first lane of each substrate RNA. Complexes were separated on 4% native polyacrylamide gels. (E) Graphic representation of EMSA data. The percentage of bound RNA (input RNA − unbound RNA/input RNA × 100) is plotted against the concentration of recombinant ELAV. Results are from at least three experiments. Error bars indicate standard deviations.
FIG. 5.
FIG. 5.
ELAV complex boundary experiment with substrate RNA pA2-I. (A and B) Partially hydrolyzed 5′ (A) or 3′ (B) 32P-labeled substrate RNA pA2-I was extracted from gel-purified ELAV complexes and analyzed on 5% denaturing gels. Markers are partial RNase A (lanes 1) or RNase A and T1 (lanes 2) digests of pA2-I RNA or a single-nucleotide ladder of pA2-I RNA (lanes 3). Positions within the substrate RNA are indicated on the left, and sequence landmarks are shown on the right. Binding by the ELAV complex is indicated by a black bar for 5′-labeled substrate RNA and by a gray bar for 3′-labeled substrate RNA on the right sides of A and B, respectively. (C) Schematic summary of the boundary experiment. RNAs bound by the ELAV complex are indicated as black bars for 5′-labeled substrate RNA above the pA2-I sequence and as gray bars for 3′-labeled substrate RNA below the pA2-I sequence. The starts of the bars indicate the minimal lengths of the RNA required for complex formation. Poly(U) motifs are in boldface. Vector sequence is indicated by dashed lines and is shown in Materials and Methods. Note that 5′- and 3′-selected RNAs largely overlap and thus indicate absence of a unique sequence element for binding.
FIG. 6.
FIG. 6.
Evolutionary comparison of the ELAV binding site in ewg intron 6. (A) The DNA sequence from the AAUAAA of pA2 up to exon I was obtained from a series of Drosophila species from one of the main lines in the melanogaster species group and aligned. Sequence landmarks of corresponding transcripts are shown above the alignment above a consensus sequence deduced as majority (e.g., ≥7 from 13). Consensus strength is indicated by colored bars (red, 12 or 13 identical from 13; orange, 10 or 11 identical from 13; green, 8 or 9 identical from 13; light blue, 6 or 7 identical from 13; and dark blue, 4 or 5 identical from 13). R, purine; Y, pyrimidine; and W, A or T. Ns or spaces are shown as dots. Consensus nucleotides are in yellow in the alignment, and conserved AU4 motifs are in yellow in the consensus sequence. If the consensus nucleotide is a purine, then the minor portion is in green. Similarly, orange is used for pyrimidines and blue for A/T. Taxonomical relations are shown at the 3′ end of the sequence. (B) Tree of molecular relations based on nucleotide substitutions (generated with MegAlign, DNAstar version 5.06).
FIG. 7.
FIG. 7.
Inhibition of 3′ end formation at pA2 depends on ELAV binding at multiple and spaced AU4-6 motifs in vivo. (A) Schematic of the last ewg intron 6 and 3′ UTR with splicing and polyadenylation choices as present with HA and VSV tags in the tcgER rescue reporter construct (top) (55). The two major isoforms are those with intron 6 spliced from H to J in the presence of ELAV (solid line) or with polyadenylation in intron 6 at pA2 in the absence of ELAV. ewg probes used for RPA are shown in the middle. A magnification of the ELAV binding region is shown at the bottom, depicting polyadenylation site pA2 and part of exon I as well as AU4-6 motif and spacer mutations introduced into tcgER reporter transgenes. (B) RPA of RNA prepared from 20 third-instar larval brains from wild-type and AU4-6 motif mutants of ewg rescue reporter construct transgenes in a wild-type elav background. After purification, RNA was run on a 6% denaturing gel. Protected fragments are indicated at the right, and protected fragments from endogenous ewg transcripts are indicated by asterisks. The fragment marked with x derives from exon J of the transgene (judged by its accumulation following the usage of intronic polyadenylation sites). The loading control protects a fragment from Appl. Quantification of protected fragments from intron 6 (unspliced) to spliced transcripts is shown at the bottom of the panel. Additional lines with independent insertions from each construct gave comparable results. (C) Splicing of ewg intron 6 analyzed by quantitative RT-PCR (primers 6F and VSV-R) with RNA extracted from third-instar eye imaginal disks from wild-type and AU4-6 motif mutants of ewg reporter construct transgenes in a wild-type elav background. For total transcripts a 5′ fragment of the reporter transcript was amplified with primers eeF and eeR (55). Uniformly 32P-dCTP labeled PCR products were separated on 8% polyacrylamide gels. (D) Splicing of ewg intron 6 analyzed by quantitative RT-PCR with RNA extracted from third-instar eye imaginal disks from wild-type and spacer mutants of ewg reporter construct transgenes in a wild-type elav background. For total transcripts a 5′ fragment of the reporter transcript was amplified with primers eeF and eeR. Uniformly 32P-dCTP labeled PCR products were separated on 8% polyacrylamide gels. Quantification from three independent insertions is shown as means at the bottom.

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