Dysregulation of a family of short noncoding RNAs, tsRNAs, in human cancer

Abstract

Chronic lymphocytic leukemia (CLL) is the most common human leukemia, and transgenic mouse studies indicate that activation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene is a contributing event in the pathogenesis of the aggressive form of this disease. While studying the regulation of TCL1 expression, we identified the microRNA cluster miR-4521/3676 and discovered that these two microRNAs are associated with tRNA sequences and that this region can produce two small RNAs, members of a recently identified class of small noncoding RNAs, tRNA-derived small RNAs (tsRNAs). We further proved that miR-3676 and miR-4521 are tsRNAs using Northern blot analysis. We found that, like ts-3676, ts-4521 is down-regulated and mutated in CLL. Analysis of lung cancer samples revealed that both ts-3676 and ts-4521 are down-regulated and mutated in patient tumor samples. Because tsRNAs are similar in nature to piRNAs [P-element-induced wimpy testis (Piwi)-interacting small RNAs], we investigated whether ts-3676 and ts-4521 can interact with Piwi proteins and found these two tsRNAs in complexes containing Piwi-like protein 2 (PIWIL2). To determine whether other tsRNAs are involved in cancer, we generated a custom microarray chip containing 120 tsRNAs 16 bp or more in size. Microarray hybridization experiments revealed tsRNA signatures in CLL and lung cancer, indicating that, like microRNAs, tsRNAs may have an oncogenic and/or tumor-suppressor function in hematopoietic malignancies and solid tumors. Thus, our results show that tsRNAs are dysregulated in human cancer.

Keywords: ts-3676; ts-4521; tsRNAs.

Fig. 1.

Ts-3676 and ts-4521 at 17p13. (A) TsRNAs are produced from the 3′ ends of pre-tRNAs. (B) Genomic structure of ts-3676 and ts-4521.

Fig. 2.

The mir-3676/4521 locus produces tsRNAs. (A) Northern blot analysis of endogenous ts-4521 and commercially purchased miR-4521 (Ambion-4521). (B) Sequence of ts-4521 and ts-3676 and the probes used to prove that both small these RNAs are tsRNAs. (C and D) Northern blots showing that small RNAs 4521 (C) and 3676 (D) are tsRNAs. HEK-293 cells were transfected with the indicated constructs. NT, not transfected.

Fig. 3.

ts-3676 and ts-4521 interact with PiwiL2. (Upper) Enrichment of indicated RNA molecules in the complexes of indicated proteins vs. IgG control. N/E, no enrichment. (Lower) Expression of Ago1, Ago2, and PiwiL2 in these experiments, determined by the Western blot using anti-Omni antibody.

Fig. 4.

ts-3676 and ts-4521 are down-regulated in CLL and lung cancer. (A) Down-regulation of ts-4521 expression in four cytogenetic groups of CLL. (B and C) Expression of ts-4521 (B) and ts-3676 (C) in 17 paired lung cancer samples (T) and matched normal lung tissues (N).

Fig. S1.

Expression of ts-4521 in four different cytogenetic groups of CLL and CD19⁺ B-cell controls. Averages are shown as black bars.

Fig. S2.

ts-3676 (red bars) and ts-4521 (blue bars) show similar expression patterns in CLL.

Fig. S3.

Colony assay. H1299 and A549 lung cancer cell lines were transfected with the expression vector expressing ts-3676 or empty vector. Five thousand transfected cells were plated in each well; 5 d later, colonies were colored with brilliant blue.

Fig. 5.

TsRNA signatures in CLL and lung cancer. (A) Aggressive CLL vs. normal CD19⁺ B cells (negative values indicate down-regulation in CLL). (B) Aggressive CLL vs. indolent CLL (negative values indicate down-regulation in aggressive CLL). (C) Indolent CLL vs. normal CD19⁺ B cells (negative values indicate down-regulation in CLL). (D) Lung cancer cells vs. normal lung (negative values indicate down-regulation in lung cancer).

Fig. S4.

Specificity of the tsRNA microarray chip. HEK 293 cells were transfected with pEGFP-3676 (Left) or pEGRP-4521 (Right). Overexpression of ts-3676 (Upper Left) and ts-4521 (Lower Right) confirmed the specificity of the chip.