Characterizing medaka visual features using a high-throughput optomotor response assay.

Medaka fish (Oryzias latipes) is a powerful model to study genetics underlying the developmental and functional traits of the vertebrate visual system. We established a simple and high-throughput optomotor response (OMR) assay utilizing medaka larvae to study visual functions including visual acuity and contrast sensitivity. Our assay presents multiple adjustable stripes in motion to individual fish in a linear arena. For that the OMR assay employs a tablet display and the Fish Stripes software to adjust speed, width, color, and contrast of the stripes. Our results demonstrated that optomotor responses were robustly induced by black and white stripes presented from below in the linear-pool-arena. We detected robust strain specific differences in the OMR when comparing long established medaka inbred strains. We observed an interesting training effect upon the initial exposure of larvae to thick stripes, which allowed them to better respond to narrower stripes. The OMR setup and protocol presented here provide an efficient tool for quantitative phenotype mapping, addressing visual acuity, trainability of cortical neurons, color sensitivity, locomotor response, retinal regeneration and others. Our open-source setup presented here provides a crucial prerequisite for ultimately addressing the genetic basis of those processes.

Dissecting telomerase RNA structural heterogeneity in living human cells with DMS-MaPseq.

Telomerase is a specialized reverse transcriptase that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-containing pseudoknot (t/PK) and the three-way junction (CR4/5). These two hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are thus essential for telomerase catalytic activity. Here, we probe the structure of hTR in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and ensemble deconvolution analysis. Unexpectedly, approximately 15% of the steady state population of hTR has a CR4/5 conformation lacking features required for hTERT binding. Mutagenesis demonstrates that stabilization of the alternative CR4/5 conformation is detrimental to telomerase assembly and activity. We propose that this misfolded portion of the cellular hTR pool is either slowly refolded or degraded. Thus, kinetic traps for RNA folding that have been so well-studied in vitro may also present barriers for assembly of ribonucleoprotein complexes in vivo.