tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma

Abstract

Sequencing studies from several model systems have suggested that diverse and abundant small RNAs may be derived from tRNA, but the function of these molecules remains undefined. Here, we demonstrate that one such tRNA-derived fragment, cloned from human mature B cells and designated CU1276, in fact 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. Expression of CU1276 is abundant in normal germinal center B cells but absent in germinal center-derived lymphomas, suggesting a role in the pathogenesis of this disease. Furthermore, CU1276 represses endogenous RPA1, an essential gene involved in many aspects of DNA dynamics, and consequently, expression of this tRNA-derived microRNA in a lymphoma cell line suppresses proliferation and modulates the molecular response to DNA damage. These results establish that functionally active microRNAs can be derived from tRNA, thus defining a class of genetic entities with potentially important biological roles.

Fig. 1.

CU1276 is a Dicer-dependent tRNA fragment expressed in mature B cells. (A) Cloning frequency of CU1276 from naïve (N), GC, and memory (M) B cells purified from human tonsil, and from the Burkitt’s lymphoma cell line Ramos (RA). Data are derived from analysis of previously published small RNA libraries (6). (B) FASTA sequences of annotated human tRNAs with perfect match to the CU1276 small RNA. CU1276 sequence is highlighted in bold. (C) Northern blot analysis of total RNA from GC B cells, Ramos, and 293T cells transiently transfected with empty vector or a vector encoding for the Gly(GCC) chr1.tRNA68. The three primary bands correspond to the 22-nt tRNA fragment CU1276, the 74-nt mature tRNA, and a high molecular weight tRNA primary transcript. See also Fig. S1. (D) Western blot and Northern blot analyses of 293T cells stably expressing control shRNA (shCTRL) or a pool of three Dicer-targeting shRNA (shDicer), transiently transfected with empty or Gly(GCC) chr1.tRNA68 vector. Ethidium bromide (EtBr) staining and immunoblotting for ACTB were used as loading controls for Northern blot and Western blot, respectively.

Fig. 2.

CU1276 is bound by all four human Argonaute proteins and functions as a miRNA. qRT-PCR of CU1276 in pan-Ago and control immunoprecipitation (IP) fractions from RIVA cells (A), qRT-PCR of CU1276 in HA IP fractions from 293T cells transiently expressing equivalent levels of HA-tagged EGFP, AGO1, AGO2, AGO3, or AGO4 proteins (B), and qRT-PCR of miR-16 and CU1276 in IPs from 293T cells expressing HA-tagged EGFP, or increasing amounts of HA-tagged AGO1 (C). In all qRT-PCR graphs, values were normalized to 5s rRNA and plotted relative to control IP levels. Error bars represent the SD of triplicate qRT-PCRs. (D) Antisense 3′UTR reporter activity in response to CU1276, with or without exogenous AGO2 expression. Firefly luciferase values were normalized to a Renilla luciferase control and plotted relative to reporter cotransfected with empty vector; error bars represent the SD of two independent experiments, each performed in duplicate.

Fig. 3.

CU1276 is down-regulated in lymphoma cell lines and primary biopsies. (A) qRT-PCR of CU1276 in GC samples and B-cell lymphoma lines, including Burkitt lymphoma (BL), activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and GC-like diffuse large B cell lymphoma (GCB-DLBCL); qRT-PCR levels were normalized to RNU66 and graphed relative to GC1. Error bars represent the SD of triplicate PCR reactions (see also Fig. S2). (B) CU1276 counts from deep sequencing of small RNA libraries from purified GC (n = 4) and primary biopsies of DLBCL (n = 25). DLBCL includes ABC-DLBCL (n = 13) and GCB-DLBCL (n = 12) subtypes.

Fig. 4.

CU1276 directly represses RPA1. (A Upper) Schematic representation of the region in the 3′UTR of RPA1 targeted by CU1276. The mutations introduced into this region are highlighted in gray. (A Lower) RPA1 3′UTR reporter activity in response to CU1276 expression. Firefly luciferase values were normalized to a Renilla luciferase control, and plotted relative to reporter cotransfected with empty vector; error bars represent the SD of seven independent experiments, each performed in duplicate. The reporter is significantly repressed by either Gly(GCC) chr1.tRNA68- (Student’s t test, P = 3.9e-5) or hairpin-mediated delivery (Student’s t test, P = 8.2e-5) of CU1276. (B) Western blot analyses of RPA1 and RPA2 from 293T cells transiently transfected with empty, Gly(GCC) chr1.tRNA68-expressing-, or CU1276 hairpin-expressing vector, and from stable lines of the Burkitt lymphoma cell line P3HR1 engineered to inducibly express GFP (empty) or GFP plus CU1276 (CU1276). GFP indicates successful doxycycline induction of vector. ACTB was used as loading control. Images are representative of four independent experiments each; see also Fig. S4. (C) Western blot analysis and graphical quantification of RPA1, RPA2, and DICER1, with ACTB used as loading control, from normal GC B cells and a panel of GC-derived lymphoma cell lines, including BL, activated B-cell-like diffuse large B cell lymphoma (ABC-DLBCL), and GCB-DLBCL. The GC sample was obtained by pooling cells from two independent donors. All samples are identical to those used for CU1276 expression analysis in Fig. 3A.

Fig. 5.

CU1276 modulates proliferation and DNA damage signaling in an RPA1-dependent manner. Growth curves of P3HR1 stable cell lines containing bidirectional, doxycycline-inducible vectors expressing GFP alone (blue line), GFP plus the CU1276 hairpin (red line), or RPA1 plus the CU1276 hairpin (orange line) (A Upper), and corresponding Western blot analysis of RPA1 protein levels from these cell lines, with ACTB used as loading control (A Lower). Growth curve data are compiled from eight independent experiments, with each genotype represented by four independently derived bulk populations. Error bars represent the 95% confidence intervals of each cell type, calculated according to a normal distribution. CU1276 expression is sufficient to significantly reduce cellular proliferation relative to the GFP control at 96 h (Student’s t test, *P = 1.8e-3). At 96 h, RPA1 rescue restores growth completely to wild-type levels. (B) Western blot analysis of RPA1, total H2AFX, and γH2AFX in etoposide-treated control cells and cells expressing CU1276. ACTB was used as loading control. Image is representative of three independent experiments, for which average γH2AFX quantifications are indicated in bar chart format. Error bars represent the SD of three independent experiments. (C) Western blot analysis of control cells, cells expressing CU1276, and cells simultaneously expressing CU1276 and exogenous RPA1. Restoration of RPA1 protein levels rescues CU1276-mediated sensitization of H2AFX phosphorylation upon etoposide treatment. ACTB was used as loading control. Image is representative of three independent experiments, for which average γH2AFX quantifications are indicated in bar chart format. Error bars represent SDs.