A novel class of small RNAs, tRNA-derived RNA fragments (tRFs), has been identified. These tRFs are derived from precise processing at the 5' or 3' end of mature or precursor tRNAs, forming three series: tRF-5, tRF-3, and tRF-1. tRFs are the second most abundant class of small RNAs after miRNAs. They are not random by-products of tRNA degradation but are specifically structured and have distinct biological roles. tRF-1001, derived from the 3' end of a Ser-TGA tRNA precursor transcript, is highly expressed in cancer cell lines and is associated with cell proliferation. Knockdown of tRF-1001 using siRNA impaired cell proliferation, with cells accumulating in the G2 phase, which was reversed by reintroducing a synthetic 2'-O-methyl tRF-1001 oligoribonucleotide. tRF-1001 is generated in the cytoplasm by the tRNA 3'-endonuclease ELAC2, a prostate cancer susceptibility gene. These findings suggest that tRFs are a novel class of small RNAs with specific functions in cellular processes. The study highlights the importance of tRFs in biological functions and their potential roles in disease.A novel class of small RNAs, tRNA-derived RNA fragments (tRFs), has been identified. These tRFs are derived from precise processing at the 5' or 3' end of mature or precursor tRNAs, forming three series: tRF-5, tRF-3, and tRF-1. tRFs are the second most abundant class of small RNAs after miRNAs. They are not random by-products of tRNA degradation but are specifically structured and have distinct biological roles. tRF-1001, derived from the 3' end of a Ser-TGA tRNA precursor transcript, is highly expressed in cancer cell lines and is associated with cell proliferation. Knockdown of tRF-1001 using siRNA impaired cell proliferation, with cells accumulating in the G2 phase, which was reversed by reintroducing a synthetic 2'-O-methyl tRF-1001 oligoribonucleotide. tRF-1001 is generated in the cytoplasm by the tRNA 3'-endonuclease ELAC2, a prostate cancer susceptibility gene. These findings suggest that tRFs are a novel class of small RNAs with specific functions in cellular processes. The study highlights the importance of tRFs in biological functions and their potential roles in disease.