Dysregulation of different classes of tRNA fragments in chronic lymphocytic leukemia

Abstract

Chronic lymphocytic leukemia (CLL) is the most common human leukemia, and dysregulation of tRNA-derived short noncoding RNA (tsRNA) (tRF-1) expression is an accompanying event in the development of this disease. tsRNAs are fragments originating from the 3' end of tRNA precursors and do not contain mature tRNA sequences. In contrast to tsRNAs, mature tRFs (tRF-3s, tRF-5s, and internal tRFs) are produced from mature tRNA sequences and are redundant fragments. We investigated tsRNA expression in CLL and determined tsRNA signatures in indolent CLL and aggressive CLL vs. normal B cells. We noticed that both ts-43 and ts-44 are derived from distinct genes of pre-tRNA^His, and are down-regulated in CLL 3- to 5-fold vs. normal B cells. Thus, we investigated expression levels of tRF-5 fragments from tRNA^His in CLL samples and healthy controls, and determined that such fragments are down-regulated by 5-fold in CLLs vs. normal controls. Given these results, we investigated the expression of all mature tRFs in CLLs vs. normal controls. We found a drastic dysregulation of the expression of mature tRFs in CLL. In aggressive CLL, for the top 15 up-regulated fragments, linear fold change varied from 2,053- to 622-fold. For the top 15 down-regulated fragments in CLL, linear fold change varied from 314- to 52-fold. In addition, 964 mature tRFs were up-regulated at least 2-fold in CLL, while 701 fragments were down-regulated at least 2-fold. Similar results were obtained for indolent CLL. Our results suggest that mature tRFs may have oncogenic and/or tumor suppressor function in CLL.

Keywords: tRFs; tRNA fragments; tsRNAs.

Fig. 1.

Down-regulation of tsRNA expression in CLL. (A–C) Real-time RT-PCR analysis shows a significant decrease of ts-36, ts-42, and ts-70 expression in patient samples (indolent CLL, IDs 1473 and 1212; aggressive CLL, IDs 1493 and 1653) compared with normal B cells. One-tailed Wilcoxon rank-sum test was applied for statistical analysis to all 3 experiments. *P < 0.05 was considered statistically significant (P = 0.02857 for each of the 3 tsRNAs considered). Controls are derived from 3 distinct healthy individuals. (D) Graphic representation of the methylation status of the CpG islands inside the ts-42 promoter region (human build GRCh38/hg38). Methylated cytosine residues in the representative chromatograms are indicated by arrows. (E) The table shows the methylation rate for selected genomic sites inside the ts-42 promoter region. We used commercial genomic DNA (Clontech), the HEK-293TN cell line, and normal B cells as controls. Error bars indicate the SD for the 3 replicates.

Fig. 2.

Down-regulation of tRF-5 expression from tRNA^His in CLL. One-tailed Wilcoxon rank-sum test was applied for statistical analysis. P < 0.05 was considered statistically significant (with a P value of 0.001651 for indolent CLL vs. normal B cells, and a P value of 0.0006553 for aggressive CLL vs. normal B cells). Annotations for **P < 0.01 are provided accordingly. Controls are derived from 8 distinct healthy individuals. Error bars indicate the SD for the 3 replicates.

Fig. 3.

Dysregulation of selected mature tRFs in CLL by real-time RT-PCR. We selected 10 tRFs dysregulated in CLL for custom real-time RT-PCR. (Top) Expression of 5 mature tRFs up-regulated in CLL. (Bottom) Expression of 5 mature tRFs down-regulated in CLL as well. The single CLL sample belongs to the group of indolent CLLs, and its ID is 1477. The normal B cell sample is 6139. Error bars indicate the SD for the 3 replicates.