Noncanonical cytoplasmic poly(A) polymerases regulate RNA levels, alternative RNA processing, and synaptic plasticity but not hippocampal-dependent behaviours

Abstract

Noncanonical poly(A) polymerases are frequently tethered to mRNA 3' untranslated regions and regulate poly(A) tail length and resulting translation. In the brain, one such poly(A) polymerase is Gld2, which is anchored to mRNA by the RNA-binding protein CPEB1 to control local translation at postsynaptic regions. Depletion of CPEB1 or Gld2 from the mouse hippocampus results in a deficit in long-term potentiation (LTP), but only depletion of CPEB1 alters animal behaviour. To test whether a related enzyme, Gld4, compensates for the lack of Gld2, we separately or simultaneously depleted both proteins from hippocampal area CA1 and again found little change in animal behaviour, but observed a deficit in LTP as well as an increase in long-term depression (LTD), two forms of protein synthesis-dependent synaptic plasticity. RNA-seq data from Gld2, Gld4, and Gld2/Gld4-depleted hippocampus show widespread changes in steady state RNA levels, alternative splicing, and alternative poly(A) site selection. Many of the RNAs subject to these alterations encode proteins that mediate synaptic function, suggesting a molecular foundation for impaired synaptic plasticity.

Keywords: Noncanonical poly(A) polymerase; RNA-seq; alternative RNA processing; animal behaviour; synaptic plasticity.

Figure 1.

Effects of noncanonical poly(A) polymerase depletion from the hippocampus. A. AAV9 vectors expressing a scrambled (i.e., non-specific) control sequence or shRNAs for Gld2 or Gld4 in various combinations were injected bilaterally into the mouse hippocampus in 3 separate batches. Batch 1 animals were analysed for various behaviours as were batch 2 animals, but in this case, the animals were sacrificed and the hippocampus processed for RNA-seq. Batch 3 animals were processed for electrophysiology. B. Batch 1 and 2 animals injected with scrambled (n = 19), Gld2 shRNA (n = 19) (KD, or knockdown), Gld4 shRNA (n = 19) or a combination of Gld2 and Gld4 shRNAs (n = 18) were examined on an elevated plus maze for time spent at the closed or open arms or at the junction. C. Batch 1 and 2 animals were subjected to an open field test and assessed for total distance moved, outer distance, centre distance, duration in the centre or duration on the outer periphery (scrambled n = 14; Gld2 shRNA, n = 14; Gld4 shRNA n = 14; Gld2/4 shRNA n = 13). D. Batch 1 animals were assessed for novel object recognition (scrambled n = 11; Gld2 shRNA n = 7; Gld4 shRNA n = 7; Gld2/4 shRNA n = 5). E. Batch 1 animals were examined in a T maze for day 1 and 2 (scrambled n = 9; Gld2 shRNA n = 8; Gld4 shRNA n = 9; Gld2/4 shRNA n = 8). F. Batch 1 animals were assessed in a passive avoidance chamber during training and 24 hours later (scrambled n = 8; Gld2 shRNA n = 10; Gld4 shRNA n = 10; Gld2/4 shRNA n = 9). G. Batch 2 animals were examined for marble burying capacity (scrambled n = 10; Gld2 shRNA n = 9; Gld4 shRNA n = 9; ld2/4 shRNA n = 9). * p < 0.05, one-way non-parametric ANOVA. H. Batch 2 animas were assessed for social behaviour in a 3-chamber sociality task; time in the stranger side, the middle, or the empty side was determined (scrambled n = 10; Gld2 shRNA n = 9; Gld4 shRNA n = 9; Gld2/4 shRNA n = 9)

Figure 2.

Effect of noncanonical poly(A) polymerase depletion on plasticity of hippocampal Schafer collateral CA1 synapses. AAV9 vectors expressing a scrambled control sequence or shRNAs for Gld2 and Gld4 were injected bilaterally into the mouse hippocampus. A. Stimulus–response relationship of Schaffer Collateral to CA1 (SC-CA1) synaptic responses from Control scrambled and Gld2/4 shRNA mice. No effect is found on the relationship between presynaptic stimulus strength and the magnitude of postsynaptic response. B. Decreased TBS-LTP at SC-CA1 synapses in Gld2/4 shRNA compared to control (scrambled shRNA-injected) mice. Insert: Decreased envelope of TBS-induced depolarization (average of the nine bursts of the TBS induction protocol) in Gld2/4 shRNA compared to control scrambled mice. C. Enhanced glycine-LTD at SC-CA1 synapses in Gld2/4 shRNA compared to control scrambled mice. Data are expressed as mean ± s.e.m (n = 5 mice). The fEPSP slopes acquired during distinct time intervals after LTP or LTD induction (i.e., 20–30, 60–90, 150–180 min, etc.) were averaged for each group and compared using analysis of variance (MICROCAL ORIGIN statistical tool, Microcal Software Inc.). *, p < 0.05; **, p < 0.01

Figure 3.

Noncanonical poly(A) polymerases regulate steady state RNA levels. A. Volcano plots demonstrating RNAs that are elevated (red) or reduced (blue) following injection of scrambled (NS), Gld2 shRNA, Gld4 shRNA, or Gld2 and Gld4 (Gld2_Gld4) shRNAs. Multiple pair-wise comparisons are shown; the number of RNAs up or down regulated are indicated (all changes >1.5 fold; padj <0.05). B. Venn diagrams illustrating the extent of overlap for RNAs up and down regulated in multiple pair-wise comparisons. C. Cluster analysis presented as a heat map of RNAs up and down regulated (cluster 1, 1430 RNAs; cluster 2, 634 RNAs; cluster 3, 980 RNAs; cluster 4, 88 RNAs). The colour bar at right indicates log2 fold change. Biologic triplicates for each condition are presented

Figure 4.

Noncanonical poly(A) polymerases regulate exon alternative RNA processing. Multiple pair-wise comparisons of skipped exons following noncanonical poly(A) polymerase depletion. Gld2 vs scrambled control (NS) (No change, NC, grey 3986 RNAs, skipped exons (blue), 669; included exons (red) 440); Gld4 vs NS (NC (grey), 5530; skipped exons (blue) 270; included exons (red) 270; Gld2/4 vs NS (NC, grey 3982; skipped exons (blue) 638; included exons (red) 391); Gld2/4 vs Gld2 (NC, grey 4396; skipped exons (blue) 385; included exons (red) 352); Gld2/4 vs Gld4 (NC, grey 4319; skipped exons (blue) 635; included exons (red) 421); Gld4 vs Gld2 (NC, grey 43,239; skipped exons (blue) 518; included exons (red) 704). PSI, percent spliced in

Figure 5.

Noncanonical poly(A) polymerases regulate alternative poly(A) site selection (APA). A. Pair-wise comparisons of RNA with lengthened (i.e., using a distal poly(A) site, red) or shortened (i.e., using a proximal poly(A) site, blue) 3ʹUTRs. The number of RNAs in each category are indicated (NC, no change; Sh, shortened; Ln, lengthened). Plots are log2 ratios of constitutive 3ʹ UTR (cUTR) vs. alternative 3ʹ UTR (aUTR). B. GO analysis of RNAs with long 3ʹUTRs following Gld2 depletion compared to scrambled (NS). Also shown is GO analysis of RNAs with short 3ʹUTRs following Gld4 depletion compared to scrambled control (NS). All are padj<0.05. Gene ratio refers to the total of differentially processed RNAs in the give GO term