Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution.

The breadth and importance of RNA modifications are growing rapidly as modified ribonucleotides can impact the sequence, structure, function, stability, and fate of RNAs and their interactions with other molecules. Therefore, knowing cellular RNA modifications at single-base resolution could provide important information regarding cell status and fate. A current major limitation is the lack of methods that allow the reproducible profiling of multiple modifications simultaneously, transcriptome-wide and at single-base resolution. Here we developed RBS-Seq, a modification of RNA bisulfite sequencing that enables the sensitive and simultaneous detection of m5C, Ψ, and m1A at single-base resolution transcriptome-wide. With RBS-Seq, m5C and m1A are accurately detected based on known signature base mismatches and are detected here simultaneously along with Ψ sites that show a 1-2 base deletion. Structural analyses revealed the mechanism underlying the deletion signature, which involves Ψ-monobisulfite adduction, heat-induced ribose ring opening, and Mg2+-assisted reorientation, causing base-skipping during cDNA synthesis. Detection of each of these modifications through a unique chemistry allows high-precision mapping of all three modifications within the same RNA molecule, enabling covariation studies. Application of RBS-Seq on HeLa RNA revealed almost all known m5C, m1A, and ψ sites in tRNAs and rRNAs and provided hundreds of new m5C and Ψ sites in noncoding RNAs and mRNAs. However, our results diverge greatly from earlier work, suggesting ∼10-fold fewer m5C sites in noncoding and coding RNAs and the absence of substantial m1A in mRNAs. Taken together, the approaches and refined datasets in this work will greatly enable future epitranscriptome studies.

Experimental Approaches for Target Profiling of RNA Cytosine Methyltransferases.

RNA cytosine methyltransferases (m(5)C-RMTs) constitute an important class of RNA-modifying enzymes, methylating specific cytosines within particular RNA targets in both coding and noncoding RNAs. Almost all organisms express at least one m(5)C-RMT, and vertebrates often express different types or variants of m(5)C-RMTs in different cell types. Deletion or mutation of particular m(5)C-RMTs is connected to severe pathological manifestations ranging from developmental defects to infertility and mental retardation. Some m(5)C-RMTs show spatiotemporal patterns of expression and activity requiring careful experimental design for their analysis in order to capture their context-dependent targets. An essential step for understanding the functions of both the enzymes and the modified cytosines is defining the one-to-one connection between particular m(5)C-RMTs and their target cytosines. Recent technological and methodological advances have provided researchers with new tools to comprehensively explore RNA cytosine methylation and methyltransferases. Here, we describe three complementary approaches applicable for both discovery and validation of candidate target sites of specific m(5)C-RMTs.

Custom designing therapeutic cancer vaccines: delivery of immunostimulatory molecule adjuvants by protein transfer.

Attempts to create vaccines for humans against invading pathogens such as viruses and bacteria have met with tremendous success. The process of developing vaccines against these pathogens is greatly aided by the fact that they contain antigens that are entirely foreign to humans. Although the knowledge and strategies developed for designing vaccines against these microbes may be of use in developing cancer vaccines, the poor antigenicity and immunosuppressive ability of cancers pose major hurdles to vaccine development. Established tumors have not only withstood immune screening and selection pressure, making them poor stimulators of an immune response, but have also adapted mechanisms to continue evading immune surveillance by creating an immunosuppressive environment. Also, genetic differences in immune responses to an antigen among individuals result in an antigenic profile that varies from patient to patient. Cancers bear such great similarities to normal cells in the body that, on a molecular level, the differences between cancerous and non-cancerous cells are minor. Therefore, developing vaccines which use the host's own tumor tissues carries the risk of breaking tolerance to self-antigens that are present in the tumor tissue. Vaccination strategies that will optimally stimulate the immune system against tumor specific antigens under immunosuppressive conditions need to be developed. In practical terms, this calls for a method by which therapeutic vaccines may be custom-designed to treat cancers case by case. Ex vivo manipulation of dendritic cells and gene transfer of immunostimulatory molecules in ex vivo expanded tumors are being tested in both experimental models and also in human clinical trials. Some of them have met with limited success. Emerging technologies such as protein transfer, which make it possible to express immunostimulatory molecules on tumor cell membranes, offer the means to develop efficient tumor vaccines that are simple and fast, while being easy to store and administer in human patients. Progress in these techniques will move the cancer vaccine field a step closer towards realizing custom designed cancer vaccines in human clinical settings.