Scatter Irradiation of Rat Brain Triggers Sex- and Brain Region-Specific Changes in the Expression of Non-Coding RNA Fragments

Abstract

Non-coding RNA fragments (ncRFs) are small RNA fragments processed from non-coding RNAs (ncRNAs). ncRFs have various functions and are commonly tissue-specific, and their processing is altered by exposure to stress. Information about ncRFs in the brain is scarce. Recently, we reported the brain region-specific and sex-specific expression of ncRNAs and their processing into ncRFs. Here, we analyzed the expression of ncRFs in the frontal cortex (FC), hippocampus (HIP), and cerebellum (CER) of male and female rats exposed to scatter radiation. We found multiple brain region- and sex-specific changes in response to scatter radiation. Specifically, we observed decreased miRNA expression and the increased expression of ra-ncRNA reads in HIP and CER, as well as an increased number of mtR-NA-associated reads in HIP. We also observed the appearance of sense-intronic ncRNAs-in females, in HIP and FC, and in males, in CER. In this work, we also show that tRNA-GlyGCC and tRNA-GlyCCC are most frequently processed to tRFs, in CER in females, as compared to males. An analysis of the targeted pathways revealed that tRFs and snoRFs in scatter radiation samples mapped to genes in several pathways associated with various neuronal functions. While in HIP and CER these pathways were underrepresented, in FC, they were overrepresented. Such changes may play an important role in pathologies that develop in response to scatter radiation, the effect known as "radio-brain", and may in part explain the sex-specific differences observed in animals and humans exposed to radiation and scatter radiation.

Keywords: cerebellum; frontal cortex; hippocampus; ncRNA fragments; non-coding RNA; sex-specific; tRFs.

Figure 1

Mapping of reads to various ncRNAs in the FC, HIP, and CER of male and female rats. In (A) (female) and (B) (male), the y-axis shows the number of mapped reads; numbers over the bars show the percentage of mapped reads. In (C) (female) and (D) (male), the y-axis shows the mapping to a specific chromosome out of 1.0, representing 100%. In all cases, the x-axis shows the brain regions for the control (Ct) and scatter-irradiate (SC) animals.

Figure 2

Mapping of sequence reads to various ncRNAs in the cerebellum (CER) (A), frontal cortex (FC) (B), and hippocampus (HIP) (C) of control and scatter-irradiated male and female rats. The y-axis shows the mapping out of 1.0, representing 100%, while the x-axis shows various types of ncRNAs.

Figure 3

Differences in the size of the reads mapping to different ncRNAs in the FC, HIP, and CER regions of control and scatter radiation-exposed male and female rats. (A) Read size in the FC of control and scatter-irradiated males and females; (B) Read size in the HIP of control and scatter-irradiated males and females; (C) Read size in the CER of control and scatter-irradiated males and females. The y-axis shows the size of the reads, while the x-axis lists various types of ncRNAs. The bottom and top of the rectangle indicate the first and third quartiles, respectively. The lower and upper ends of the vertical line extending outside the rectangle represent the minimum and maximum, respectively. The thick horizontal line inside the rectangle is the median, and the circle beyond the rectangle displays an outlier.

Figure 4

Number of rRF (A), snoRF (B), tRF (C), and snRF (D) reads in the CER, FC, and HIP of control and scatter-irradiated male and female rats. “Ct_M”—control male, “SR_M”—scatter radiation-exposed males, “Ct_F”—control females, “SR_F”—scatter radiation-exposed males. The y-axis shows the prorated read number, while the x-axis shows the group of animals.

Figure 5

Size of rRF (A), snoRF (B), tRF (C), and snRF (D) reads in the CER, FC, and HIP of control and scatter-irradiated male and female rats. “Ct_M”—control male, “SR_M”—scatter radiation-exposed males, “Ct_F”—control females, “SR_F”—scatter radiation-exposed males. The y-axis shows the size of the reads, while the x-axis shows the group of animals.

Figure 6

Comparison of the number of tRF reads mapping to different tRNAs in control and scatter radiation-exposed groups of males and females in the CER (A), FC (C), and HIP (E) brain regions. Since reads mapping to Gly were predominant, we generated another set of figures omitting Gly reads—CER (B), FC (D), and HIP (F). The y-axis shows the number of reads mapping to tRNA. The x-axis shows the tRNA to which the reads mapped by type.

Figure 7

The enrichment of tRFs processed from tRNAs in the CER (A), HIP (B), and FC (C) of control and SC animals. “HIP_Male”—hippocampus of male rats; “HIP_Female”—hippocampus of female rats; “FC_Male”—frontal cortex of male rats; “FC_Female”—frontal cortex of female rats; “CER_Male”—cerebellum of male rats; “CER_Female”—cerebellum of female rats; “Ct”—control; “SC”—scatter radiation. The y-axis shows specific tRNA and tRF-5′. The x-axis shows specific classes of tRNAs. When the tRF peak is larger than the tRNA peak, there is an enrichment, while when it is lower, there is underrepresentation.

Figure 8

snoRF-5′ enrichment from snoRNAs in the CER (A), HIP (B), and FC (C) of control and SC animals. “HIP_Male”—hippocampus of male rats; “HIP_Female”—hippocampus of female rats; “FC_Male”—frontal cortex of male rats; “FC_Female”—frontal cortex of female rats; “CER_Male”—cerebellum of male rats; “CER_Female”—cerebellum of female rats; “Ct”—control; “SC”—scatter radiation. The y-axis shows specific snoRNA and snoRF-3 ratios. The x-axis shows specific snoRNA/snoRF. When the snoRF peak is larger than the snoRNA peak, there is an enrichment, while when it is lower, there is underrepresentation.

Figure 9

snoRF-3′ enrichment from snoRNAs in the CER (A), HIP (B), and FC (C) of control and SC animals. “HIP_Male”—hippocampus of male rats; “HIP_Female”—hippocampus of female rats; “FC_Male”—frontal cortex of male rats; “FC_Female”—frontal cortex of female rats; “CER_Male”—cerebellum of male rats; “CER_Female”—cerebellum of female rats; “Ct”—control; “SC”—scatter radiation. The y-axis shows specific snoRNA and snoRF-3 ratios. The x-axis shows specific snoRNA/snoRF. When the snoRF peak is larger than the snoRNA peak, there is an enrichment, while when it is lower, there is underrepresentation.

Figure 10

Venn diagrams of overlapping target genes: (A) as analyzed by miRDB and target pathways; (B) as analyzed by DAVID of tRFs in brain regions of control and scatter-irradiated male and female rats.

Figure 11

Venn diagrams of overlapping target genes: (A) as analyzed by miRDB and target pathways; (B) as analyzed by DAVID of rRFs in brain regions of control and scatter-irradiated male and female rats.

Figure 12

Venn diagrams of overlapping target genes: (A) as analyzed by miRDB and target pathways; (B) as analyzed by DAVID of snoRFs in brain regions of control and scatter-irradiated male and female rats.

Figure 13

Venn diagrams of overlapping target genes (analyzed by miRDB) of snRFs in brain regions of control and scatter-irradiated male and female rats.

Figure 14

Venn diagrams of overlapping target genes (analyzed by miRDB) of tRFs (A) and snoRFs (B) in brain regions of control and scatter-irradiated male and female rats after Benjamini correction.