Girish C. Shukla

miR 488* inhibits androgen receptor expression in prostate carcinoma cells

Androgen receptor (AR) is a ligand‐dependent transcription factor, which plays a significant role in prostate carcinogenesis. Blockade of AR and its ligand, androgen is the basis for the treatment of prostate cancer (PCa). Nevertheless, a modest increase in the critical levels of AR mRNA and corresponding protein is sufficient for the development of resistance to antiandrogen therapy. A strategy to further downregulate AR mRNA and protein expression in combination with antiandrogen therapy may prevent or delay the development of androgen‐independent PCa. Recent studies show that microRNAs (miRNAs) perform tumor suppressor functions in various cancers. In this study, we demonstrate that the overexpression of miR 488* downregulates the transcriptional activity of AR and inhibits the endogenous AR protein production in both androgen‐dependent and androgen‐independent PCa cells. In addition, miR 488* blocks the proliferation and enhances the apoptosis of PCa cells. Our data indicate that miR 488* targets AR and is a potential modulator of AR mediated signaling. Our findings provide insight for utilizing miRNAs as novel therapeutics to target AR in PCa.

A Boost for the Emerging Field of RNA Nanotechnology Report on the First International Conference on RNA Nanotechnology

This Nano Focus article highlights recent advances in RNA nanotechnology as presented at the First International Conference of RNA Nanotechnology and Therapeutics, which took place in Cleveland, OH, USA (October 23–25, 2010) (http://www.eng.uc.edu/nanomedicine/RNA2010/), chaired by Peixuan Guo and co-chaired by David Rueda and Scott Tenenbaum. The conference was the first of its kind to bring together more than 30 invited speakers in the frontier of RNA nanotechnology from France, Sweden, South Korea, China, and throughout the United States to discuss RNA nanotechnology and its applications. It provided a platform for researchers from academia, government, and the pharmaceutical industry to share existing knowledge, vision, technology, and challenges in the field and promoted collaborations among researchers interested in advancing this emerging scientific discipline. The meeting covered a range of topics, including biophysical and single-molecule approaches for characterization of RNA nanostructures; structure studies on RNA nanoparticles by chemical or biochemical approaches, computation, prediction, and modeling of RNA nanoparticle structures; methods for the assembly of RNA nanoparticles; chemistry for RNA synthesis, conjugation, and labeling; and application of RNA nanoparticles in therapeutics. A special invited talk on the well-established principles of DNA nanotechnology was arranged to provide models for RNA nanotechnology. An Administrator from National Institutes of Health (NIH) National Cancer Institute (NCI) Alliance for Nanotechnology in Cancer discussed the current nanocancer research directions and future funding opportunities at NCI. As indicated by the feedback received from the invited speakers and the meeting participants, this meeting was extremely successful, exciting, and informative, covering many groundbreaking findings, pioneering ideas, and novel discoveries.

A Catalytically Active Group II Intron Domain 5 Can Function in the U12-Dependent Spliceosome

Both spliceosomal and self-splicing group II introns require the function of similar small, metal binding RNA stem-loop elements located in U6 or U6atac snRNAs of the spliceosome or domain 5 (D5) of group II introns. Here we report that two different D5 elements can functionally replace the U6atac snRNA stem-loop in an in vivo splicing assay. For efficient function in vivo, a single base pair from the upper helical section of the D5 sequence had to be removed. Introducing the equivalent base pair deletion into the D5 element of a group II intron reduced but did not eliminate self-splicing activity. Our results strengthen the case that these RNA elements play similar roles in the catalytic centers of both the spliceosome and a self-splicing ribozyme.

Intrinsic expression of host genes and intronic miRNAs in prostate carcinoma cells

BackgroundRecent data show aberrant and altered expression of regulatory noncoding micro (mi) RNAs in prostate cancer (PCa). A large number of miRNAs are encoded in organized intronic clusters within many protein coding genes. While expression profiling studies of miRNAs are common place, little is known about the host gene and their resident miRNAs coordinated expression in PCa cells. Furthermore, whether expression of a subset of miRNAs is distinct in androgen-responsive and androgen-independent cells is not clear. Here we have examined the expression of mature miRNAs of miR 17–92, miR 106b-25 and miR 23b-24 clusters along with their host genes C13orf25, MCM7 and AMPO respectively in PCa cell lines.ResultsThe expression profiling of miRNAs and host genes was performed in androgen-sensitive MDA PCa 2b and LNCaP as well as in androgen-refractory PC-3 and DU 145 cell culture models of PCa. No significant correlation between the miRNA expression and the intrinsic hormone-responsive property of PCa cells was observed. Androgen-sensitive MDA PCa 2b cells exhibited the highest level of expression of most miRNAs studied in this report. We found significant expression variations between host genes and their resident miRNAs. The expressions of C13orf25 and miR 17–92 cluster as well as MCM7 and miR 106b-25 cluster did not reveal statistically significant correlation, thus suggesting that host genes and resident miRNAs may be expressed independent of each other.ConclusionOur results suggest that miRNA expression profiles may not predict intrinsic hormone-sensitive environment of PCa cells. More importantly, our data indicate the possibility of additional novel mechanisms for intronic miRNA processing in PCa cells.

The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop

The U6 spliceosomal snRNA forms an intramolecular stem-loop structure during spliceosome assembly that is required for splicing and is proposed to be at or near the catalytic center of the spliceosome. U6atac snRNA, the analog of U6 snRNA used in the U12-dependent splicing of the minor class of spliceosomal introns, contains a similar stem-loop whose structure but not sequence is conserved between humans and plants. To determine if the U6 and U6atac stem-loops are functionally analogous, the stem-loops from human and budding yeast U6 snRNAs were substituted for the U6atac snRNA structure and tested in an in vivo genetic suppression assay. Both chimeric U6/U6atac snRNA constructs were active for splicing in vivo. In contrast, several mutations of the native U6atac stem-loop that either delete putatively unpaired residues or disrupt the putative stem regions were inactive for splicing. Compensatory mutations that are expected to restore base pairing within the stem regions restored splicing activity. However, other mutants that retained base pairing potential were inactive, suggesting that functional groups within the stem regions may contribute to function. These results show that the U6atac snRNA stem-loop structure is required for in vivo splicing within the U12-dependent spliceosome and that its role is likely to be similar to that of the U6 snRNA intramolecular stem-loop.

Housekeeping gene selection advisory: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are targets of miR-644a.

Results of overexpression or downregulation of a microRNA (miRNA) on its target mRNA expression are often validated by reverse-transcription and quantitative PCR analysis using an appropriate housekeeping gene as an internal control. The possible direct or indirect effects of a miRNA on the expression of housekeeping genes are often overlooked. Among many housekeeping genes, expressions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin have been used extensively for normalization of gene expression data. Here, we show that GAPDH and β-actin are direct targets of miR-644a. Our data demonstrate the unsuitability of GAPDH and β-actin as internal controls in miR-644a functional studies and emphasize the need to carefully consider the choice of a reference gene in miRNA experiments.

A revised model for U4atac/U6atac snRNA base pairing.

The discovery of a second type of spliceosomal intron in the genomes of many multicellular eukaryotes raised the exciting possibility of comparing the structural and functional features of the two splicing systems with the hope of shedding light on important features of each (Tarn & Steitz, 1997)+ This hope was amply fulfilled with the discovery of the parallel roles played in the two systems by the respective sets of spliceosomal snRNAs+ The initial identification of functions for the previously known U11 and U12 snRNAs (Hall & Padgett, 1994; Tarn & Steitz, 1996b) was magnified by the discovery of the novel U4atac and U6atac snRNAs (Tarn & Steitz, 1996a)+ The similarities of these latter two snRNAs to the U4 and U6 snRNAs of the major splicing system were apparent and led to detailed comparisons of the features of these RNAs and their interactions (Tarn & Steitz, 1996a)+ For both the U4/U6 pair and the U4atac/

miR-377-dependent BCL-xL regulation drives chemotherapeutic resistance in B-cell lymphoid malignancies

BackgroundBCL-xL is an anti-apoptotic BCL-2 family protein that inhibits apoptosis and is overexpressed in many cancers. We have reported that acquired resistance to the BCL-2 inhibitor ABT-199 (venetoclax) is associated with increased BCL-xL expression. Yet, how BCL-xL mediates chemoresistance in hematopoietic malignancies is not clear. This finding may help in design of new strategies for therapeutic intervention to overcome acquired chemoresistance mediated by BCL-xL.ResultsWe now show that the increased BCL-xL expression was inversely correlated with that of miR-377 in ABT-199-resistant cells. This finding was also extended to a panel of B-cell lymphoid lines and primary chronic lymphocytic leukemia (CLL) cells. miR-377 suppressed BCL-xL expression by recognizing two binding sites in the BCL-xL 3’-UTR. Mutation of these two miR-377 consensus-binding sites completely abolished its regulatory effect. Expression of a miR-377 mimic downregulated BCL-xL protein expression and significantly increased apoptotic cell death. Expression of a miR-377 inhibitor restored BCL-xL protein expression and limited cell death caused by the hypomethylating agent 5-azacytidine. Thus, miR-377-dependent BCL-xL regulation drives acquired therapeutic resistance to ABT-199. We further show that CLL patients who received a diverse array of chemotherapy regimens also had significantly higher BCL-xL and lower miR377 expression, indicating that exposure to chemotherapy might trigger transcriptional silencing of miR-377, which results in high levels of BCL-xL. Importantly, CLL patients with high BCL-xL/low miR-377 expression had an advanced tumor stage. Moreover, the high BCL-xL expression correlated with short treatment-free survival in 76 CLL patients. miR-377 is located at 14q32 in the DLK1-DIO3 region, which encodes the largest tumor suppressor miRNA cluster in humans. Examination of five additional 14q32 miRNAs revealed that the majority were significantly down-regulated in most CLL patients as well as in ABT-199-resistant cell lines. Remarkably, four of these miRNAs had significantly decreased expression in chemotherapy-treated CLL patients as compared to those untreated. These findings indicate a reduced expression of multiple miRNAs that may reflect a global silencing of this miRNA cluster in therapy-resistant lymphoid cells.ConclusionsThese findings reveal a novel mechanism by which down-regulation of miR-377 increases BCL-xL expression, promoting chemotherapy resistance in B-cell lymphoid malignancies.

U4 small nuclear RNA can function in both the major and minor spliceosomes

U4 small nuclear RNA (snRNA) and U6 snRNA form a base-paired di-snRNP complex that is essential for pre-mRNA splicing of the major class of metazoan nuclear introns. The functionally analogous but highly diverged U4atac and U6atac snRNAs form a similar complex that is involved in splicing of the minor class of introns. Previous results with mutants of U6atac in which a substructure was replaced by the analogous structure from U6 snRNA suggested that wild-type U4 snRNA might be able to interact productively with the mutant U6atac snRNA. Here we show that a mutant U4 snRNA designed to base pair with a mutant U6atac snRNA can activate U12-dependent splicing when coexpressed in an in vivo genetic suppression assay. This genetic interaction could also be demonstrated in an in vitro crosslinking assay. These results show that a U4/U6atac di-snRNP can correctly splice a U12-dependent intron and suggest that the specificity for spliceosome type resides in the U6 and U6atac small nuclear ribonucleoproteins. Further experiments suggest that expression of a mutant U4 snRNA that can bind to wild-type U6atac snRNA alters the specificity of some splice sites, providing an evolutionary rationale for maintaining two U4-like snRNAs.

Functionally important structural elements of U12 snRNA

U12 snRNA is analogous to U2 snRNA of the U2-dependent spliceosome and is essential for the splicing of U12-dependent introns in metazoan cells. The essential region of U12 snRNA, which base pairs to the branch site of minor class introns is well characterized. However, other regions which are outside of the branch site base pairing region are not yet characterized and the requirement of these structures in U12-dependent splicing is not clear. U12 snRNA is predicted to form an intricate secondary structure containing several stem–loops and single-stranded regions. Using a previously characterized branch site genetic suppression assay, we generated second-site mutations in the suppressor U12 snRNA to investigate the in vivo requirement of structural elements in U12-dependent splicing. Our results show that stem–loop IIa is essential and required for in vivo splicing. Interestingly, an evolutionarily conserved stem–loop IIb is dispensable for splicing. We also show that stem–loop III, which binds to a p65 RNA binding protein of the U11-U12 di.snRNP complex, is essential for in vivo splicing. The data validate the existence of proposed stem–loops of U12 snRNA and provide experimental support for individual secondary structures.