A functional role for transfer RNA-derived microRNAs in human B cells
Sequencing studies performed in recent decades have revealed that the cells of most eukaryotic organisms express a diverse repertoire of small RNAs. Genetic and biochemical investigation of these molecules has demonstrated that many small RNAs, most notably the microRNA subclass, possess the capacity to influence the expression of other genes, adding substantial complexity to our understanding of genetic regulatory networks. In the years since their initial discovery, microRNAs have been implicated in nearly every aspect of metazoan biology, including medically relevant processes such as the development of mammalian immune cells and the oncogenic transformation of such cells into leukemias and lymphomas. Even in these well-studied systems, however, the function of only a small fraction of microRNAs is understood, and novel RNAs may yet remain undiscovered. Thus, in order to better define the microRNA landscape of both normal mature B cells and their oncogenic counterparts, we undertook sequencing of their small RNA transcriptomes. In addition to microRNAs, these studies unexpectedly identified a class of RNA fragments whose sequences matched to transfer RNA, but whose size distribution resembled that of microRNAs. Deep sequencing of small RNAs from a panel of normal mature B cells and B cell lymphomas reveals that these cell types express thousands of unique transfer RNA-derived fragments, with highly distinct expression profiles in each biological subtype. We hypothesized that these fragments might be derived from direct processing of tRNA but nonetheless function as microRNA, and sought to experimentally characterize one representative sequence of this class, cloned from human mature B cells and designated CU1276. The resulting data demonstrate that CU1276 does indeed possesses the functional characteristics of a microRNA, including a DICER1-dependent biogenesis, physical association with Argonaute proteins, and the ability to repress mRNA transcripts in a sequence-specific manner. Specifically, CU1276 represses endogenous expression of RPA1, a gene with critical functions in many aspects of DNA dynamics, including replication and repair. CU1276 is abundantly expressed in normal mature B cells but strongly downregulated in B cell-derived lymphomas, while its target, RPA1, is overexpressed in lymphomas. Furthermore, enforced expression of CU1276 in a lymphoma cell line results in an RPA1-dependent impairment of both proliferation and DNA damage repair. These results suggest that relief from CU1276-mediated repression of RPA1 may confer a selective advantage to lymphoma cells, and they shed light on a possible regulatory role for transfer RNA-derived microRNA in the in the maturation of normal B cells. Taken together with published data, these results suggest that in a broad spectrum of organisms and tissues, transfer RNAs act as a previously unrecognized substrate for the biogenesis of microRNA, with substantial implications for the future study of small RNA-mediated gene regulation.
Networked Digital Library of Theses and Dissertations