A comprehensive repertoire of tRNA-derived fragments in prostate cancer

Abstract

Prostate cancer (PCa) is the most common cancer among men in developed countries. Although its genetic background is thoroughly investigated, rather little is known about the role of small non-coding RNAs (sncRNA) in this disease. tRNA-derived fragments (tRFs) represent a new class of sncRNAs, which are present in a broad range of species and have been reported to play a role in several cellular processes. Here, we analyzed the expression of tRFs in fresh frozen patient samples derived from normal adjacent prostate and different stages of PCa by RNA-sequencing. We identified 598 unique tRFs, many of which are deregulated in cancer samples when compared to normal adjacent tissue. Most of the identified tRFs are derived from the 5'- and 3'-ends of mature cytosolic tRNAs, but we also found tRFs produced from other parts of tRNAs, including pre-tRNA trailers and leaders, as well as tRFs from mitochondrial tRNAs. The 5'-derived tRFs comprise the most abundant class of tRFs in general and represent the major class among upregulated tRFs. The 3'-derived tRFs types are dominant among downregulated tRFs in PCa. We validated the expression of three tRFs using qPCR. The ratio of tRFs derived from tRNALysCTT and tRNAPheGAA emerged as a good indicator of progression-free survival and a candidate prognostic marker. This study provides a systematic catalogue of tRFs and their dysregulation in PCa and can serve as the basis for further research on the biomarker potential and functional roles of tRFs in this disease.

Keywords: RNA-sequencing; biomarker; non-coding RNA; prostate cancer (PCa); tRNA-derived fragments (tRFs).

Figure 1. tRF types in prostate cancer

A. Heatmap showing the total read counts mapped to individual tRNA isotypes in three study groups: NAP, normal adjacent prostate; PCa, prostate cancer group (consisting of 6 different sample pools, the average value is shown); LN_PCa, lymph node metastasis. The color and its corresponding value in log₁₀ scale are depicted on the right. B. tRF length as based on the read abundance (orange bars) and uniqueness (dotted line). C. Graph depicting the locations of mapped tRFs on the sequences of mature tRNAs. Full-length tRNA sequences are aligned to the middle using the anticodon position. tRFs mapped to these tRNAs are depicted as grey bars which relative abundance per particular tRNA is reflected by the color intensity (light grey, low abundance; black, high abundance). tRNAs with only one mapped tRF are clustered at the top, tRNAs with two mapped tRFs in the middle and tRNAs with multiple mapped tRFs are at the bottom. D-E. Start (blue line) and end (orange line) positions of tRFs on the mature tRNA sequence. Relative abundance of each tRF start and end based on the uniqueness (D) or abundance (E) is shown. F. An illustration of various tRF types and their approximate location on the secondary structure of tRNA. G. Relative proportions of each tRF types in our dataset as based on the uniqueness (% of unique independent reads) or abundance (% of total number of reads).

Figure 2. Differentially expressed tRFs in prostate cancer

Plotted are normalized read-count values of each tRF in the normal adjacent prostate versus various stages of prostate cancer. The baseline value for tRFs that are not expressed is 1. Full lines represent 4-fold change borderlines. Colored points represent significantly changed tRFs (Kal's Z-test on proportions, Bonferroni corrected p-values, p < 0.05) labeled as 5’-derived (magenta) and 3’-derived (green) tRFs. tRFs with the 3’-nucleotide at a position ≤40 on the precursor tRNA sequence are considered as 5′-derived. tRFs with the start nucleotide at a position ≥30 on the precursor tRNA sequence are considered as 3′-derived. tRFs that do not fall into either of these two categories are shown in blue. Positions of tRF-310, tRF-315, and tRF-389 are indicated as an example of three differentially expressed tRFs. The graph at the bottom right corner summarizes the total number of differentially expressed tRFs per group. The amount of tRFs with ≥4-fold differential expression are indicated in dark orange (upregulated) or dark blue (downregulated) color.

Figure 3. Frequency of tRF types among differentially expressed tRFs

A-B. Start (blue line) and end (orange line) positions of tRFs on the mature tRNA sequence and the proportions of each tRF type for 72 upregulated (A) and 24 downregulated (B) tRFs. C-H. Graphs showing the exact positions of 6 selected tRFs (shown in color) and other tRFs (shown in grey) on their tRNA precursors. The x-axis represents the position on the precursor sequence in nucleotides. The y-axis represents summed read-counts per sample group. The expression level per group is indicated by different color (see the legend at the bottom). Expression levels of other tRFs are shown as means across PCa groups.

Figure 4. qPCR validation of tRF-544, tRF-315, and tRF-562

A-C. RNA expression of tRF-544 (A), tRF-315 (B), and tRF-562 (C) in cohorts of clinical samples obtained from Erasmus MC (cohort 1) and Tampere University Hospital (cohort 2). Mean values are indicated by a red line. D. Ratio of tRF-315 (derived from tRNA^LysCTT) to tRF-544 (derived from tRNA^PheGAA). E. Progression-free survival curves of cohort 1 and cohort 2 based on the tRF-315/tRF544 ratio. Legend: NAP, normal adjacent prostate; PCa, prostate cancer; PCa cur (PCa cured), patients with no disease recurrence after radical prostatectomy; PCa recur (PCa recurrent), patients with biochemical recurrence or metastatic progression after radical prostatectomy; Gl <7, Gleason score <7; Gl ≥7, Gleason score 7, 8 or 9; pT2, pathological stage 2; pT3, pathological stage 3; *P-value ≤0.05; **P-value ≤0.01; ***P-value ≤0.001.