Pri-miR-17-92a transcript folds into a tertiary structure and autoregulates its processing

Abstract

MicroRNAs control gene expression either by RNA transcript degradation or translational repression. Expressions of miRNAs are highly regulated in tissues, disruption of which leads to disease. How this regulation is achieved and maintained is still largely unknown. MiRNAs that reside on clustered or polycistronic transcripts represent a more complex case where individual miRNAs from a cluster are processed with different efficiencies despite being cotranscribed. To shed light on the regulatory mechanisms that might be operating in these cases, we considered the long polycistronic primary miRNA transcript pri-miR-17-92a that contains six miRNAs with diverse functions. The six miRNA domains on this cluster are differentially processed to produce varying amounts of resultant mature miRNAs in different tissues. How this is achieved is not known. We show, using various biochemical and biophysical methods coupled with mutational studies, that pri-miR-17-92a adopts a specific three-dimensional architecture that poses a kinetic barrier to its own processing. This tertiary structure could create suboptimal protein recognition sites on the pri-miRNA cluster due to higher-order structure formation.

Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages

“…Macrophages lacking the Mirc1/Mir17-92 cluster were obtained from Mirc1/Mir17-92 ¡/¡ floxed mice. 36 Since a complete knockout of the cluster is embryonic lethal, these mice were obtained by breeding a Lyz2/LysM-cre mouse with a Mirc1/Mir17-92 ¡/¡ floxed mouse, thus, knocking out the Mirc1/Mir17-92 cluster specifically in the myeloid lineage. 27 The protein lysates of WT and Mirc1/Mir17-92 ¡/¡ macrophages were analyzed by western blot using specific autophagy antibodies.…”

Degradation dynamics of microRNAs revealed by a novel pulse-chase approach

“…Our theoretical calculation assumes that processing is much faster than cell division (k 2 >> α), a simplification that did not hold for complex miRNA clusters, where processing efficiency plays an important role in dictating the levels of each miRNA contained in the cluster. A paradigmatic example is the miR-17-92 cluster, comprising six individual miRNAs processed with different efficiencies, despite being cotranscribed due to a particular tertiary structure of the pri-miRNA (Chakraborty et al 2012). We have to underline that low efficiency maturation also affects half-life calculations by metabolic labeling.…”

Fingerprinting the junctions of RNA structure by an open-paddlewheel diruthenium compound

“…Hydroxyl radical cleavage intensities were determined as in Lozano et al (2014), and normalized to a scale from 0 to 1.5, where 1.0 is defined as the average intensity of highly reactive residues (Chakraborty et al 2012). Nucleotides with reactivity values lower than half the mean reactivity are defined as solvent-inaccessible.…”