Ribosomal Proteins Rpl22 and Rpl22l1 Control Morphogenesis by Regulating Pre-mRNA Splicing

Abstract

Most ribosomal proteins (RP) are regarded as essential, static components that contribute only to ribosome biogenesis and protein synthesis. However, emerging evidence suggests that RNA-binding RP are dynamic and can influence cellular processes by performing "extraribosomal," regulatory functions involving binding to select critical target mRNAs. We report here that the RP, Rpl22, and its highly homologous paralog Rpl22-Like1 (Rpl22l1 or Like1) play critical, extraribosomal roles in embryogenesis. Indeed, they antagonistically control morphogenesis through developmentally regulated localization to the nucleus, where they modulate splicing of the pre-mRNA encoding smad2, an essential transcriptional effector of Nodal/TGF-β signaling. During gastrulation, Rpl22 binds to intronic sequences of smad2 pre-mRNA and induces exon 9 skipping in cooperation with hnRNP-A1. This action is opposed by its paralog, Like1, which promotes exon 9 inclusion in the mature transcript. The nuclear roles of these RP in controlling morphogenesis represent a fundamentally different and paradigm-shifting mode of action for RP.

Keywords: Rpl22; Rpl22l1; Smad2; extraribosomal function; gastrulation; hnRNP-A1; morphogenesis; paralog; pre-mRNA splicing; ribosomal protein.

Figure 1. Opposing roles of RP paralogs, Rpl22 and Like1, in regulating gastrulation

(A,B) One-cell stage zebrafish embryos were injected with translational-blocking Like1-A-MO (2ng), L22-A-MO (6ng) or both (D-MOs), following which effects on gastrulation were assessed. The images of embryos represent lateral views at 10hpf. The red and black lines indicate the anterior and posterior ends of the body axis, respectively. The angle (θ), which defines the degree of extension, was measured between the red arrow and dashed black line, and is represented graphically as the mean ± standard deviation (S.D.). Control (black); Like1 MO (red); Rpl22 MO (yellow); and double-morphants (D-MO; green). Triplicate samples were quantified and the mean ± S.D. is depicted graphically. **, p < 0.01. (C) Imaging of notochord in Like1 morphants (10hpf) co-injected with 100pg mCherry-CAAX mRNA at the 1-cell stage. The dorsal view, anterior is at the top. The lateral notochord boundaries are indicated by the dotted yellow lines, and the width of notochord was marked by white lines. (D,D’) Expression patterns of ntl in 10hpf Like1 morphants. Red Arrows mark changes in distribution in the images representing lateral (D) and dorsal (D’) views. (E,F) sox32 and lefty1 expression in Like1 morphants. Red Arrows indicated changes in expression or distibution. (E) 75%-epiboly stage, dorsal view. (F) lateral view, 16hpf. (G-I) Phospho-Smad2 and total-Smad2 were assessed in 10hpf (G), 16hpf (H), and 4.7hpf (I) Like1 morphants by immunoblotting. All results are representative of at least 3 experiments performed. See also Figure S1.

Figure 2. The gastrulation defects in Like1 morphants result from skipping of smad2 exon 9

(A,B) RT-PCR analysis of smad2 mRNA in Like1, Rpl22 and double morphants (D-MOs). smad2 mRNA was evaluated by RT-PCR using primers (black arrowheads) amplifying the sequences between exons 5 to 10. (C) Like1 knockdown causes skipping of smad2 exon 9. Sequence analysis of the smaller smad2 mRNA species caused by Like1 knockdown (red arrow in panel A). (D) Quantitative RT-PCR analysis of the relative expression of intact smad2 mRNA (indicated by left panel, blue arrow). The blue and red lines identify the position of real-time primers employed to detect intact smad2 mRNA. Triplicate samples were quantified and the mean ± S.D. is depicted graphically. p-values are indicated. All results are representative of at least 3 experiments performed. See also Figure S2.

Figure 3. The C&E defects in Like1 morphants can be rescued by re-establishing smad2 signaling

(A) Schematic of the morpholino employed to induce exon9 skipping (S2-i8e9-MO). (B) Immunoblotting of detergent extracts reveals a reduction in total and phospho-Smad2 protein expression in the S2-i8e9-morphants. (C–E) S2-i8e9-MO induction of smad2 mis-splicing phenocopied the C&E defects caused by Like1 knockdown, as indicated by altered morphology (C, red arrow) and alterations in ntl/hgg1/dlx3b and lefty1 expression and distribution, as measured by in situ hybridization (D, 10hpf, red arrows, anterior dorsal view; E, 16hpf, lateral view). (F–H) mRNA encoding constitutively activated smad2 (Ca-Smad2, 20pg) was utilized for injection alone or co-injected with Like1-A-MO. Embryo morphology (F) as well as the abnormal distribution of ntl (G, red arrows) and myod1 (H, red arrows) at 10hpf, can be rescued by ectopic expression of Ca-Smad2. All embryos are dorsal view with the anterior on top at 10hpf. All results are representative of at least 3 experiments performed. See also Figure S3.

Figure 4. The regulation of smad2 pre-mRNA splicing during gastrulation by Rpl22 and Like1 is associated with their retention in the nucleus

(A,B) Subcellular location of epitope-tagged Rpl22 and Like1 at 10hpf (A) and 24hpf (B). 100pg of mRNA encoding HA-zRpl22 (HA/L22) and HA-zLike1 (HA/L1) was co-injected with mRNA encoding mCherry-CAAX into 1-cell stage embryos and visualized by HA antibody immunostaining. mCherry-CAAX marked the cell membrane and DAPI marked the nucleus. Red scale bar =10 µm. (C) RT-PCR detection of smad2 mis-splicing. Following Like1 MO injection, smad2 mis-splicing was assessed by RT-PCR in 10hpf and 24hpf Like1 morphants. All results are representative of at least 3 experiments performed. See also Figure S4.

Figure 5. Effect of Like1-deficiency on Smad2 splicing during murine gastrulation

(A) Molecular strategy for targeted deletion of Rpl22l1. A Loxp-FRT-neo resistance-FRT cassette was inserted 3’ to the first exon of Rpl22l1 and a second LoxP site was inserted 3’ to the third exon. F1 heterozygous offspring were bred to Mox2-cre mice to delete the 2^nd and 3^rd exons of Rpl22l1, disrupting expression at the genomic locus. (B,C) Strategy to genotype Rpl22l1^+/L or Rpl22l1^+/− mice. To genotype mice with LoxP sites flanking exons 2 and 3 of Rpl22l1, primers C and D were used to amplify a 223 bp or 263 bp product for wildtype and Rpl22L1-LoxP, respectively. Deletion of Rpl22l1 is genotyped with primers B and D, which amplify a 607 bp product for the WT allele and A and D, which amplify a 420 bp product for the mutant allele after cre recombination. (D) Representative Rpl22l1^−/− embryo compared to Rpl22l1^+/+ littermate control at 9.5 dpc. (E–G) Effect of Like1-deficiency on splicing of Smad2 pre-mRNA during murine gastrulation. Embryos derived from timed matings of Rpl22l1+/− mice were isolated at 6.5 dpc (mid gastrulation), genotyped as above, and analyzed by RT-PCR using the indicated primers to identify alterations in Smad2 splicing (E,F). Sequencing of the mis-spliced Smad2 species found in Rpl22l1−/− embryos revealed that it represented a species in which exon 6 was fused directly to exon 9, eliminating exons 7 and 8 (G). All results are representative of at least 3 experiments performed.

Figure 6. Common features of mRNA targets mis-spliced in Like1 morphants

(A) smad2 exon9 skipping detected by RNA-Seq. Alignment of RNA-seq reads to the genome reveals exclusion of smad2 exon9 in 10hpf Like1 morphants (blue box). (B) Gene ontology analysis of transcripts affected by Like1 knockdown from RNA-seq analysis. Significant gene ontology terms (p < 0.05) are depicted as a bar graph with the p values represented as –log₁₀ on the X-axis. (C) Schematic illustrating the type of alternative splicing identified by RNA-Seq in 10hpf Like1 morphants. (D) Calculation of the strength of 5´ and 3´ splice sites of skipped (S) versus non-skipped (NS) exons in pre-mRNAs targeted in Like1 morphants. (E) Schematic of the consensus hairpin bound by Rpl22/Like1. (F) Schematic model of the common features of pre-mRNA targets affected by Like1 knockdown. Targets contained both a consensus Rpl22/Like1 binding motif in the preceding intron and a G-rich motif in the skipped exon. RNA-Seq analysis was performed on at least 3 independent biological replicates per condition. See also Figures S5 and S6

Figure 7

Factors through which Rpl22 and Like1 regulate smad2 alternative splicing. (A) Position of the real-time primers (red arrows) flanking the consensus Rpl22/Like1 binding site in intron 8 of smad2 pre-mRNA. (B) RNA-CLIP analysis of Rpl22/Like1 binding to smad2 pre-mRNA. Embryos injected with mRNA encoding HA tagged Rpl22, Like1, or their RNA-binding mutants (m88) were harvested at 10hpf. After light crosslinking, detergent nuclear extracts were immunoprecipitated using anti-HA antibody, and the co-precipitated RNA quantified by RT-PCR. Triplicate measurements are depicted graphically as mean ± S.D. p-values are indicated. (C) Overexpression of hnRNP-A1 (A1) mRNA induces smad2 mis-splicing. Embryos were injected with differing amounts of A1 mRNA, following which the effect on smad2 mis-splicing was determined by RT-PCR at 10hpf. The relative ratio of exon 9 skipped mRNA to intact smad2 mRNA was quantified in triplicate and depicted graphically as the mean ± S.D. (D) Genetic interaction of Rpl22 and A1. The ratio of exon9 skipped to intact smad2 was quantified in Like1 morphants in which A1 and/or Rpl22 was knocked down. The mean ± S.D of triplicate measurements was depicted graphically as in (C). (E) Physical association between Rpl22, Like1 and A1. Anti-HA immunoprecipitation (IP) and anti-Flag immunoblots (IB) were performed on detergent extracts of embryos injected with 100pg of mRNA encoding Flag-A1 and either HA-Rpl22 or HA-Like1, either before or after treatment with RNAse. All results are representative of at least 3 experiments performed. See also Figure S7.