Rat Spinal Cord Injury Associated with Spasticity Leads to Widespread Changes in the Regulation of Retained Introns

Abstract

To determine molecular changes that correlate with long-term physiological changes after spinal cord injury associated with spasticity, we used a complete transection model with an injury at sacral spinal level S2, wherein tail spasms develop in rats weeks to months post-injury. Using Illumina and nanopore sequencing, we found that from 12,266 expressed genes roughly 11% (1,342) change expression levels in the rats with spasticity. The transcription factor PU.1 (Spi-1 proto-oncogene) and several of its known regulated genes were upregulated during injury, possibly reflecting changes in cellular composition. In contrast to widespread changes in gene expression, only a few changes in alternative exon usage could be detected because of injury. There were more than 1,000 changes in retained intron usage, however. Unexpectedly, most of these retained introns have not been described yet but could be validated using direct RNA nanopore sequencing. In addition to changes from injury, our model allowed regional analysis of gene expression. Comparing the segments rostral and caudal to the injury site in naïve animals showed 525 differentially regulated genes and differential regional use of retained introns. We did not detect changes in the serotonin receptor 2C editing that were implicated previously in this spinal cord injury model. Our data suggest that regulation of intron retention of polyadenylated pre-mRNA is an important regulatory mechanism in the spinal cord under both physiological and pathophysiological conditions.

Keywords: gene expression; mRNA; pre-mRNA splicing; spasticity.

FIG. 1.

Rat spinal cord transection animal model leading to spasticity. (A) Animal model: a transection was performed between the sacral spinal cord segments S1 and S2 underneath the lumbar vertebrae L1 and L2, which leads to tail spasms after sixty days. At ∼180 days post-injury, 7 mm of spinal cord corresponding to the caudal region below the injury (BI) and 5 mm of spinal cord corresponding to the rostral region above the injury (AI) were collected. An assessment of the spasticity is shown in Supplementary Figure 1. Non-injured animals were dissected similarly, and the tissues are denoted as AN, above naïve, and BN, below naïve. (B) Representative electromyogram (EMG) showing rat tail spasticity induced by electrical stimulation. Arrow indicates the time point of stimulus. (C) Overview of the changes in gene expression. Four conditions were compared: (1) injured rostral segment with injured caudal segment; (2) injured caudal segment with naïve caudal segment; (3) naïve rostral segment with naïve caudal segment, and (4) injured rostral segment with naïve rostral segment. Numbers indicate the changes in gene expression, and arrows indicate whether upregulation or downregulation. Shaded boxes indicate the changes in exon usage. RI: retained introns. (D) Comparison of changes among the four conditions described in panel C. The Venn diagram shows the number of gene expression changes common to each condition, indicated by coloring overlaps. Arrows indicate the direction of the changes. A_Inj A_Nai: A injured compared with A naïve; B_Inj B_Nai: B injured compared with B naïve; B_Inj A_Inj: B injured compared with A injured; B_Nai A_Nai: B naïve compared with A Naïve.

FIG. 2.

Cluster analysis of overall gene expression. The unsupervised clustering of expressed genes was compared with the gene expression signatures of cell types indicated. Euclidian distance is the square root of the sum of the squared differences in gene expression. BI, below injury; BN, below naïve; AI, above injury; AN, above naïve.

FIG. 3.

Properties of retained introns (RI) regulated during spinal cord injury. (A) Structure of a RI (thick line) that can be either included (thick line) or spliced out (thin lines). (B) Position of regulated RIs in spinal cord. The position is indicated as percent of the total length of the pre-messenger ribonucleic acid. Reference is the position of rat RIs that do not undergo spinal cord regulation. (C) Length of the RIs. Reg RI: RIs differently regulated between rostral and caudal segments. Ref RI: RIs from the FAST DB database^{54, 55}; Ref const introns: constitutively spliced introns. (D–F) Splice site strength of regulated RIs expressed as maximum entropy score for the acceptor (3' splice site) and donor (5' splice site). Reg. RI: RIs regulated in spinal cord; Ref RI: all known rat RIs; Ref- constitutive: all constitutive introns.

FIG. 4.

Identification of novel retained introns in rat spinal cord. (A) Strategy to identify novel retained introns in the dataset. (B) Venn diagram showing the expression of novel retained introns and their overlap with experimental conditions in the dataset. (C–F) Comparison of the percent intron retention (PIR) of the various experimental conditions. The PIR was calculated according to panel A. (G) Association of novel regulated retained introns with metabolic pathways. The number of regulated genes is indicated by the size of the circle and the regulation through the coloring, as shown in the legend below. The color scheme shows the p value of changes in the metabolic pathways. A lower p value with a warmer color indicates a higher likelihood for significance of the annotated change. BI, below injury; BN, below naïve; AI, above injury; AN, above naïve.

FIG. 5.

Pre-messenger ribonucleic acid (pre-mRNA) processing of the serotonin receptor 2C (5HT2C). (A) Schematic overview of the 5HT2C receptor pre-mRNA. Exons are indicated by roman numbers. Exon Vb is alternatively used, depending on recognition of the distal (DS) or proximal (PS) splice site. Skipping of exon Vb leads to RNA1 encoding a truncated receptor, and its inclusion generates RNA2 that includes the full-length receptor. Exon Vb contains five editing sites (A–E) that can be deaminated from adenosine to inosine. (B) Frequency of RNA editing at given sites under the four experimental conditions, as illustrated in Figure 1C. (C) Distribution of editing variants at different experimental conditions. The sum of all variants was set to 100%. (D) Distribution of the combination of editing sites. The sequences are indicated on the left, the frequency is shown on the right, as percent of all 5HT2C reads containing the editing sites. (E) Frequency of exon Vb skipping under the four experimental conditions. BI, below injury; BN, below naïve; AI, above injury; AN, above naïve.

FIG. 6.

Model. (A) Under non-injured conditions, there are segment-specific transcription programs because of differential promoter usage in spinal cord segments. In addition, there are programs of differential retained intron regulation in genes distinct from the ones affected by promoter regulation. These introns reside in polyadenylated ribonucleic acid (RNA) that is likely retained in nuclei. (B) Injury elicits a rostral and caudal response, with the rostral response being stronger than the caudal response. (C) Spinal cord expressed numerous retained introns in polyadenylated RNA. Retained introns prevent protein formation because they likely reside in nuclei and, further, contain in frame stop codons. Their removal and subsequent messenger RNA (mRNA) translation of the mRNA depends on location and injury status.