Role of Ts-RNAs in CLL
Authors: Balatti Veronica , Palamarchuk Alexey , Rizzotto Lara , Veneziano Dario , Nigita Giovanni , Liu Chang-Gong , Rassenti Laura Z. , Kipps Thomas J. , Croce Carlo M. , Pekarsky Yuri 
Published: October 2019

Abstract
Chronic lymphocytic leukemia (CLL) is the most common human leukemia. This disease occurs in two forms, aggressive and indolent, both characterized by clonal expansion of CD5+ B-lymphocytes. Hallmarks of aggressive CLL are the high expression level of ZAP-70, the unmutated sequence of IgVH genes, and activation of the TCL1 oncogene. While investigating whether the expression of miR-3676/4521 cluster in CLL is correlated with TCL1 regulation and ZAP-70 methylation in aggressive CLLs, we found that these two microRNAs are associated with tRNA sequences and that this region produces two small RNAs, members of a novel class of small non-coding RNAs (tsRNAs).
Ts-3676/4521 cluster is located on the short arm of chromosome 17 and is co-deleted with p53 in 17p deleted CLL patients. We previously demonstrated that ts-3676 and ts-4521 are down-regulated and mutated in all CLL types. Furthermore, ts-3676 targets TCL1 3'UTR, indicating that tsRNAs can interfere with gene expression at a post-transcriptional level in a micoRNA-like manner. However, tsRNAs do not share the biogenesis mechanism of microRNAs but are cleaved from the 3' ends of pre-tRNAs by the endonuclease RNase Z and represent unique sequences starting at the 3' ends of the tRNAs and ending at a sequence of 4 consecutive T nucleotides. Thus tsRNAs are single stranded RNAs, more similar to piRNAs than miRNAs. Since piRNAs interact with Piwi proteins to affect gene methylation, we hypothesized that tsRNAs could also interact with Piwi proteins. By performing a series of RNA immunoprecipitation experiments, we verified the interaction of ts-3676 and ts-4521 with Piwi proteins, suggesting that these molecules could be involved in gene promoter methylation, hence affecting gene expression at a pre-transcriptional level in a piRNA-like manner. Since ts-3676 and ts-4521 are deregulated in CLLs, we studied if other tsRNAs are differentially regulated CLL. We retrieved the tsRNAs sequences from 3' ends of tRNA genes, obtaining a total of 120 tsRNA sequences, and designed a tsRNA microarray chip to investigate signatures of tsRNAs that can distinguish different classes of CLLs. We hybridized total RNAs extracted from 23 CLL samples (11 indolent, 12 aggressive) and 8 CD19+ B cells of healthy donors and found 17 tsRNA differentially expressed in these cohorts of samples. 9 tsRNAs are differentially expressed in aggressive CLL vs. normal B-cells, 10 tsRNAs are differentially expressed in indolent CLL vs. normal B-cells, 15 tsRNAs are differentially expressed in indolent vs. aggressive CLL. In these signatures, we identified ts-46 and ts-47 strongly downregulated in aggressive CLL, suggesting these ts-RNAs as potential tumor-suppressors. Finally, we found that ts-3676 and ts-4521 have a prognostic value for particular subgroups of CLLs. Patients with low ZAP-70 expression and mutated IgVH generally show indolent disease, while patients with high ZAP-70 expression and un-mutated IgVH show aggressive disease. However, a relatively small cohort of patients shows aggressive prognostic markers but clinically indolent disease. Our analysis revealed that expression of ts-3676 is lower in aggressive CLLs vs indolent CLLs, although, we did not test these tsRNAs in cohort of patients with aggressive prognostic markers but indolent disease. Currently there is no good marker to discriminate these patients from aggressive CLLs. Thus, we selected 8 patients with unfavorable prognostic markers but indolent disease and 12 patients with both aggressive clinical course and unfavorable prognosis markers, to test for differences in the expression level of ts-3676 and ts-4521. We found that in patients with unfavorable prognostic markers but indolent disease, expression of ts-3676 and ts-4521 was almost double of that in patients displaying both aggressive clinical course and markers.
In conclusion, we provided an evidence that tsRNAs deregulation can affect gene expression by interfering with the epigenetic machinery and/or with the post-transcriptional stability of target genes. We also identified the first tsRNAs important in CLL as new markers and/or targets for therapy: ts-3676, ts-4521, ts-46 and ts-47.

Disclosures
Kipps: Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau.