1. Developmental Biology
  2. Genetics and Genomics
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Splicing in a single neuron is coordinately controlled by RNA binding proteins and transcription factors

  1. Morgan Thompson
  2. Ryan Bixby
  3. Robert Dalton
  4. Alexa Vandenburg
  5. John A Calarco
  6. Adam D Norris  Is a corresponding author
  1. Southern Methodist University, United States
  2. University of Toronto, Canada
Research Article
Cite this article as: eLife 2019;8:e46726 doi: 10.7554/eLife.46726
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
sad-1 is alternatively spliced in single neurons.

(A) The sad-1 gene. Alternative cassette exon in blue. (B) Two-color splicing reporter schematic for sad-1 cassette exon. The cassette exon encodes a + 1 nt frameshift so that when skipped, GFP is produced with an in frame stop codon. When skipped, GFP is read out of frame without stop codons, followed by in-frame translation of RFP. (C) Whole worm fluorescent micrograph demonstrating both exon inclusion (RFP) and skipping (GFP) in many neurons, while certain neurons express only the included (ALM) of skipped (BDU) isoforms. (D–E) Higher magnification focusing on ALM and BDU neurons. (F) BDU and ALM are both paired neurons present on the left and right side of the worm. Each BDU neuron is a sister cell to an ALM neuron, derived from the same neuroblast. Scale bar represents 10 µm.

https://doi.org/10.7554/eLife.46726.003
Figure 1—figure supplement 1
Gross quantification of fraction of neurons expressing included (red), skipped (green), or both (yellow) isoforms of sad-1, in either ventral nerve cord, head, or tail.
https://doi.org/10.7554/eLife.46726.004
Figure 2 with 1 supplement
Genetic screen identifies neuronal TFs affecting sad-1 splicing in the ALM neuron.

(A) Schematic of forward genetic screen to identify regulators of sad-1 splicing in the ALM touch neuron. (B–F) ALM neurons (dashed boxes) shift from complete inclusion (RFP) to skipping (GFP) in unc-86(e1416), mec-3(e1338), or alr-1(oy42) TF mutants. Splicing phenotypes fully penetrant (n = 50 animals) (G) Previously-identified roles of the three TFs in a transcriptional cascade to control touch neuron gene expression. Scale bar represents 10 µm.

https://doi.org/10.7554/eLife.46726.005
Figure 2—figure supplement 1
TF alleles identified in genetic screen cause sad-1 splicing defects in ALM: unc-86(csb9), mec-3(csb10), alr-1(csb11).
https://doi.org/10.7554/eLife.46726.006
Figure 3 with 2 supplements
Two neuronal RBPs combinatorially control sad-1 splicing in ALM neurons.

(A) Conservation scores in the introns surrounding sad-1 exon 15, basewise phyloP26way comparison of 26 nematode species (Hubisz et al., 2011). Numbers 1–3 indicate consensus binding motifs for mbl-1 and mec-8 displayed in B-C. (B–C) cis-elements matching consensus binding motifs for mbl-1 and mec-8. (D–F) mec-8 and mbl-1 mutants both cause a partial loss of sad-1 exon inclusion. (G) mec-8; mbl-1 double mutants cause complete loss of exon inclusion, phenocopying the TF mutants. Splicing phenotypes fully penetrant (n = 50 animals) Scale bar represents 10 µm.

https://doi.org/10.7554/eLife.46726.007
Figure 3—figure supplement 1
Deletion alleles used in this study, in addition to canonical mutations and mutations identified in our forward genetic screen.

In black is a schematic of the entire gene, in red a representation of the deletion. (A) mec-8(csb22) (B) mbl-1(csb31) and mbl-1(wy560). (C) msi-1(csb24).

https://doi.org/10.7554/eLife.46726.008
Figure 3—figure supplement 2
Canonical RBP alleles of mec-8 and mbl-1 affect sad-1 splicing similarly to CRISPR deletions of mec-8 and mbl-1.

mec-8 (e398) premature stop codon mutation. mbl-1(wy560) large deletion. Splicing phenotypes fully penetrant (n = 50 animals).

https://doi.org/10.7554/eLife.46726.009
Figure 4 with 3 supplements
Neuronal TFs establish expression of both mec-8 and mbl-1 to mediate splicing of sad-1 in ALM neurons.

(A–B) A mec-8 translational GFP fosmid reporter reveals strong expression in ALM neuron (strong expression in 28/31 = 90% of animals inspected). (C) In a mec-3 TF mutant, mec-8 expression is absent specifically in ALM (no detectable expression in 43/50 = 86%, dim expression in 7/50 = 14% of animals inspected). (D) mbl-1 translational RFP fosmid reporter is expressed in ALM neuron (strong expression in 19/20 = 95% of animals inspected). (E) In a mec-3 mutant, mbl-1 expression is absent specifically in ALM (no detectable expression in 19/21 = 90%, dim expression in 2/21 = 10% of animals inspected). (F–G) Aberrant splicing of sad-1 in alr-1 TF mutants is partially rescued by over-expression of either mec-8 (6/6 animals examined) or mbl-1 (6/7 animals examined) RBPs (H–I). Scale bar represents 10 µm.

https://doi.org/10.7554/eLife.46726.010
Figure 4—figure supplement 1
Transcriptional and translational reporters for MEC-8 and MBL-1.

(A) Promoter-GFP fusion for mec-8 exhibits GFP expression in various cells but not in ALM touch neurons. Right panel represents high magnification of ALM region (dotted square in left panel). (B) Micrograph of mbl-1::RFP fosmid as in Figure 4D, demonstrating expression in numerous head neurons, motor neurons, and tail neurons.

https://doi.org/10.7554/eLife.46726.011
Figure 4—figure supplement 2
ALR-1 ChIP peaks in mec-8  and mbl-1 promoters.

ALR-1 ChIP peaks from modENCODE data suggest that ALR-1 may bind directly to the promoters of mec-8 (upper panel) and mbl-1 (lower panel), although these peaks do not immediately contain consensus binding motifs for a mouse homologue of alr-1 Arx (Berger et al., 2008). Red bars capping ChIP peaks indicate values greater than the mean +5X the standard deviation.

https://doi.org/10.7554/eLife.46726.012
Figure 4—figure supplement 3
Double heterozygotes for TFs and RBPs display moderate defects in sad-1 splicing in ALM neurons.

(A) alr-1; mbl-1 double heterozygotes. (B) mec-3; mec-8 double heterozygotes. ALM splicing phenotype fully penetrant (n = 10 animals).

https://doi.org/10.7554/eLife.46726.013
Figure 5 with 1 supplement
mbl-1 and mec-8 affect sad-1 splicing by direct interaction with sad-1 introns.

(A–B) Mutation of mbl-1 consensus sequence in sad-1 splicing reporter results in aberrant splicing in ALM neurons that phenocopies an mbl-1 mutant. (C–E) Mutation of either mec-8 binding motif, or both simultaneously, likewise results in aberrant sad-1 splicing in ALM neurons. ALM splicing phenotypes fully penetrant (n = 25 animals) Scale bar represents 10 µm.

https://doi.org/10.7554/eLife.46726.014
Figure 5—figure supplement 1
RBP overexpression does not rescue sad-1 splicing defects of cognate cis-element mutant reporters (failure to rescue in n = 20 animals for each condition).

(A) Δmec-8[1] site and (B) Δmec-8[2] site not rescued by pmec-3::mec-8 over-expression. (C) Δmbl-1 site not rescued by pmec-3::mbl-1.

https://doi.org/10.7554/eLife.46726.015
Figure 6 with 2 supplements
sad-1 splicing in motor neurons of the ventral nerve cord is controlled by mbl-1 and msi-1 RBPs.

(A–C) In wild-type worms, sad-1 is partially included in both excitatory and inhibitory motor neurons. (D) In mbl-1 mutants, exon inclusion is lost in excitatory motor neurons, but remains in inhibitory motor neurons (arrowheads). (E) msi-1 mutants lose exon inclusion in inhibitory motor neurons (arrowheads) but not in excitatory motor neurons. (F) mbl-1; msi-1 double mutants lose exon inclusion in all motor neurons in the ventral nerve cord. Splicing phenotypes in ventral nerve cord invariant (n = 15 animals) (G) Conservation scores (determined as in Figure 3A) in the introns surrounding sad-1 exon 15. Number one indicates consensus binding motifs for msi-1. (H) cis-elements matching consensus binding motifs for msi-1. Asterisk indicates anterior-posterior position of ALM neuron as anatomical reference. Splicing phenotypes fully penetrant (n = 50 animals). Scale bar represents 10 µm.

https://doi.org/10.7554/eLife.46726.016
Figure 6—figure supplement 1
MBL-1, visualized by a translational RFP fusion is expressed specifically in the excitatory cholinergic neurons of the ventral nerve cord, visualized by an unc-17::BFP promoter reporter.

(A) High-magnification micrograph of portion of the ventral nerve cord, (B) Micrograph of whole worm to demonstrate expression of MBL-1 in Cholinergic motor neurons throughout the extent of the nerve cord. (C) As in A, GABAergic neurons labeled with GFP, cholinergic neurons with BFP, demonstrating MBL-1 expression in cholinergic motor neurons but not in GABAergic motor neurons.

https://doi.org/10.7554/eLife.46726.017
Figure 6—figure supplement 2
sad-1 splicing is controlled by distinct RBPs and TFs in ventral nerve cord motor neurons.

(A) Ectopic expression of mec-8 in excitatory motor neurons (via unc-17 promoter) changes sad-1 splicing in excitatory motor neurons from partial exon inclusion to complete exon inclusion (n = 7/11 animals examined). (B) Ectopic expression of mbl-1 in inhibitory motor neurons (via unc-25 promoter) has similar effects in inhibitory motor neurons (n = 9/13 animals examined). (C) unc-3 mutants exhibit partial (12/25 worms examined) or complete (13/25 worms) loss of sad-1 exon inclusion in excitatory motor neurons. (D) unc-3 mutants exhibit partially-penetrant loss of mbl-1 expression in excitatory motor neurons (25/65 animals exhibit complete loss, 28/65 partial (example displayed), 12/65 no detectable change).

https://doi.org/10.7554/eLife.46726.018
Phenotypic convergence at the level of splicing regulation.

Different RBPs act in different neuron types to carry out the common function of mediating sad-1 exon inclusion.

https://doi.org/10.7554/eLife.46726.019

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strainunc-86(csb9)This studyJAC401Norris Lab. SMU. Dallas, TX.
Strainmec-3(csb10)This studyJAC402Norris Lab. SMU. Dallas, TX.
Strainalr-1(csb11)This studyJAC403Norris Lab. SMU. Dallas, TX.
Strainunc-86(e1416)CGC, University of MinnesotaCB1416
Strainmec-3(e1338)CGC, University of MinnesotaCB1338
Strainalr-1(oy42)CGC, University of MinnesotaPY1598
Strainmec-8(e398)CGC, University of MinnesotaCB398
Strainmec-8(csb22)This studdyJAC626Norris Lab. SMU. Dallas, TX.
Strainmbl-1(csb31)This studyJAC635Norris Lab. SMU. Dallas, TX.
Strainmbl-1(wy560)CGC, University of MinnesotaJAC002
Strainmsi-1(csb24)This studyJAC628Norris Lab. SMU. Dallas, TX.
Strainmec-8(csb22); mbl-1(wy560)This studyADN342Norris Lab. SMU. Dallas, TX.
Strainmec-8(csb22); mbl-1(csb31)This studyJAC670Norris Lab. SMU. Dallas, TX.
Strainmsi-1(csb24); mbl-1(csb31)This studyADN257Norris Lab. SMU. Dallas, TX.
Strainpmec-3::mec-8This studyADN431Norris Lab. SMU. Dallas, TX.
Strainpmec-3::mbl-1This studyADN514Norris Lab. SMU. Dallas, TX.
Strainpunc-25::mbl-1This studyADN515Norris Lab. SMU. Dallas, TX.
Strainpunc-17::mec-8This studyADN505Norris Lab. SMU. Dallas, TX.
StrainΔmbl-1 cis-element sad-1 splicing reporterThis studyADN319Norris Lab. SMU. Dallas, TX.
StrainΔmec-8[1] cis-element sad-1 splicing reporterThis studyADN364Norris Lab. SMU. Dallas, TX.
StrainΔmec-8[2] cis-element sad-1 splicing reporterThis studyADN377Norris Lab. SMU. Dallas, TX.
StrainΔmec-8[both] cis-element sad-1 splicing reporterThis studyADN333Norris Lab. SMU. Dallas, TX.
Strainsad-1 splicing reporterThis studyJAC017Norris Lab. SMU. Dallas, TX.
StrainMEC-8::GFP reporter fosmidThis studyJAC447Norris Lab. SMU. Dallas, TX.
StrainMBL-1::RFP reporter fosmidThis studyJAC576Norris Lab. SMU. Dallas, TX.
Strainpmec-8::GFPCGC, University of MinnesotaBC11068
Strainunc-3(e151)CGC, University of MinnesotaCB151

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