Endonucleolytic cleavages by RNase E generate the mature 3' termini of the three proline tRNAs in Escherichia coli.

We demonstrate here for the first time that proline tRNA 3' end maturation in Escherichia coli employs a one-step endonucleolytic pathway that does not involve any of the six 3' → 5' exonucleases (RNase T, RNase PH, RNase D, RNase BN, RNase II and polynucleotide phosphorylase [PNPase]) to generate the mature CCA terminus. Rather, RNase E is primarily responsible for the endonucleolytic removal of the entire Rho-independent transcription terminator associated with the proK, proL and proM primary transcripts by cleaving immediately downstream of the CCA determinant. In the absence of RNase E, RNase G and RNase Z are weakly able to process the proK and proM transcripts, while PNPase and RNase P are utilized in the processing of proL The terminator fragment derived from the endonucleolytic cleavage of proL transcript is degraded through a PNPase-dependent pathway. It is not clear which enzymes degrade the proK and proM terminator fragments. Our data also suggest that the mature 5' nucleotide of the proline tRNAs may be responsible for the cleavage specificity of RNase E at the 3' terminus.

Macrophages exposed to HIV viral protein disrupt lung epithelial cell integrity and mitochondrial bioenergetics via exosomal microRNA shuttling.

Antiretroviral therapy extends survival but does not eliminate HIV from its cellular reservoirs. Between immune and stromal cells in the tissue microenvironment, a dynamic intercellular communication might influence host viral immune responses via intercellular transfer of extracellular vehicles (EVs) (microvesicles, exosome, or apoptotic bodies). It is increasingly recognized that HIV-infected macrophage-secreted nucleotide-rich exosomes might play a critical role in mediating communication between macrophages and other structural cells; however, molecular mechanisms underlying cell-cell crosstalk remain unknown. Here we show that HIV-1-infected macrophages and HIV-1 proteins Tat or gp120-treated macrophages express high levels of microRNAs, including miR-23a and miR-27a. Identical miRNAs expression patterns were detected in macrophage-secreted exosomes isolated from bronchoalveolar lavage fluid of HIV transgenic rats. Tat-treated macrophage-derived exosomal miR-23a attenuated posttranscriptional modulation of key tight junction protein zonula occludens (ZO-1) 3'-UTR in epithelial cells. In parallel, exosomal miR-27a released from Tat-treated macrophages altered the mitochondrial bioenergetics of recipient lung epithelial cells by targeting peroxisome proliferator-activated receptor gamma (PPARγ), while simultaneously stimulating glycolysis. Together, exosomal miRNAs shuttle from macrophages to epithelial cells and thereby explain in part HIV-mediated lung epithelial barrier dysfunction. These studies suggest that targeting miRNAs may be of therapeutic value to enhance lung health in HIV.

Site-Selective RNA Splicing Nanozyme: DNAzyme and RtcB Conjugates on a Gold Nanoparticle.

Modifying RNA through either splicing or editing is a fundamental biological process for creating protein diversity from the same genetic code. Developing novel chemical biology tools for RNA editing has potential to transiently edit genes and to provide a better understanding of RNA biochemistry. Current techniques used to modify RNA include the use of ribozymes, adenosine deaminase, and tRNA endonucleases. Herein, we report a nanozyme that is capable of splicing virtually any RNA stem-loop. This nanozyme is comprised of a gold nanoparticle functionalized with three enzymes: two catalytic DNA strands with ribonuclease function and an RNA ligase. The nanozyme cleaves and then ligates RNA targets, performing a splicing reaction that is akin to the function of the spliceosome. Our results show that the three-enzyme reaction can remove a 19 nt segment from a 67 nt RNA loop with up to 66% efficiency. The complete nanozyme can perform the same splice reaction at 10% efficiency. These splicing nanozymes represent a new promising approach for gene manipulation that has potential for applications in living cells.