Reducing the structure bias of RNA-Seq reveals a large number of non-annotated non-coding RNA.

The study of RNA expression is the fastest growing area of genomic research. However, despite the dramatic increase in the number of sequenced transcriptomes, we still do not have accurate estimates of the number and expression levels of non-coding RNA genes. Non-coding transcripts are often overlooked due to incomplete genome annotation. In this study, we use annotation-independent detection of RNA reads generated using a reverse transcriptase with low structure bias to identify non-coding RNA. Transcripts between 20 and 500 nucleotides were filtered and crosschecked with non-coding RNA annotations revealing 111 non-annotated non-coding RNAs expressed in different cell lines and tissues. Inspecting the sequence and structural features of these transcripts indicated that 60% of these transcripts correspond to new snoRNA and tRNA-like genes. The identified genes exhibited features of their respective families in terms of structure, expression, conservation and response to depletion of interacting proteins. Together, our data reveal a new group of RNA that are difficult to detect using standard gene prediction and RNA sequencing techniques, suggesting that reliance on actual gene annotation and sequencing techniques distorts the perceived architecture of the human transcriptome.

Beyond Rule-of-five: Permeability Assessment of Semipeptidic Macrocycles.

Compounds beyond the rule-of-five are generating interest as they expand the molecular toolbox for modulating targets previously considered "undruggable". Macrocyclic peptides are an efficient class of molecules for modulating protein-protein interactions. However, predicting their permeability is difficult as they differ from small molecules. Although constrained by macrocyclization, they generally retain some conformational flexibility associated with an enhanced ability to cross biological membranes. In this study, we investigated the relationship between the structure of semi-peptidic macrocycles and their membrane permeability through structural modifications. Based on a scaffold of four amino acids and a linker, we synthesized 56 macrocycles incorporating modifications in either stereochemistry, N-methylation, or lipophilicity and assessed their passive permeability using the parallel artificial membrane permeability assay (PAMPA). Our results show that some semi-peptidic macrocycles have adequate passive permeability even with properties outside the Lipinski rule of five. We found that N-methylation in position 2 and the addition of lipophilic groups to the side chain of tyrosine led to an improvement in permeability with a decrease in tPSA and 3D-PSA. This enhancement could be attributed to the shielding effect of the lipophilic group on some regions of the macrocycle, which in turn, facilitates a favorable macrocycle conformation for permeability, suggesting some degree of chameleonic behavior.

Zinc Fingers 10 and 11 of Miz-1 undergo conformational exchange to achieve specific DNA binding.

Miz-1 (ZBTB17) is a poly-zinc finger BTB/POZ transcription factor with 12 consecutive C2H2 zinc fingers (ZFs) that binds transcriptional start sites (TSSs) to regulate the expression of genes involved in cell development and proliferation. As of now, it is not known which of the 12 consecutive ZFs are responsible for the recognition of the 24 base pair consensus sequence found at these TSSs. Evidence suggests ZFs 7-12 plays this role. We provide validation for this and describe the structural and dynamical characterization of unprecedented conformational exchange in the linker between ZFs 10 and 11. This conformational exchange uncouples ZFs 7-10 from 11 and 12 and promotes a scanning-recognition mechanism through which the two segments cooperate to bind two sub-sites at both ends of the consensus. We further show that this can result in the coiling of TSSs as part of Miz-1's mechanism of transcriptional transactivation.