Biology subjects

Palka, C.

Publications and source records attributed to Palka, C..

3 recordsLinked to original sources

bioRxiv2023

A systematic search for RNA structural switches across the human transcriptome

RNA structural switches are key regulators of gene expression in bacteria, yet their characterization in Metazoa remains limited. Here we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3UTR of the RORC transcript. In vivo DMS-MaPseq, coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated mRNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally-driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.

systems biology↗

bioRxiv2019

Folding heterogeneity in the essential human telomerase RNA three-way junction

Telomeres safeguard the genome by suppressing illicit DNA damage responses at chromosome termini. In order to compensate for incomplete DNA replication at telomeres, most continually dividing cells, including many cancers, express the telomerase ribonucleoprotein (RNP) complex. Telomerase maintains telomere length by catalyzing de novo synthesis of short DNA repeats using an internal telomerase RNA (TR) template. TRs from diverse species harbor structurally conserved domains that contribute to RNP biogenesis and function. In vertebrate TRs, the conserved regions 4 and 5 (CR4/5) fold into a three-way junction (3WJ) that binds directly to the telomerase catalytic protein subunit and is required for telomerase function. We have analyzed the structural properties of the human TR (hTR) CR4/5 domain using a combination of in vitro chemical mapping, endogenous RNP assembly assays, and single-molecule structural analysis. Our data suggest that a functionally essential stem loop within CR4/5 is not stably folded in the absence of the telomerase reverse transcriptase protein subunit in vitro. Rather, the hTR CR4/5 domain adopts a heterogeneous ensemble of conformations. RNA structural engineering intended to bias the folding landscape of the hTR CR4/5 demonstrates that a stably folded 3WJ motif is necessary but not sufficient to promote assembly of a functional RNP complex. Finally, single-molecule measurements on the hTR CR4/5 domain show that RNP assembly selects for a conformation that is not the major population in the heterogeneous free RNA ensemble, suggesting that non-canonical hTR folds may be required during telomerase biogenesis.

biochemistry↗

bioRxiv2019

Reevaluation of the RNA binding properties of the Tetrahymena thermophila telomerase reverse transcriptase N-terminal domain

Telomerase restores chromosome-capping telomeric repeats lost with each round of genome replication by DNA-templated DNA polymerases. The telomerase reverse transcriptase (TERT) N-terminal (TEN) domain is a peripheral, telomerase-specific, processivity-stimulatory addition to more conserved domains that encircle the active site cavity. Reports of ciliate, yeast, and mammalian telomerase TEN domain associations with the telomerase RNA subunit (TR) describe low affinity interactions of uncertain specificity. Unfortunately two cryo-EM structures of synthesis-paused telomerase holoenzymes lack sufficient resolution to discriminate molecular specificity of possible TR contact(s) with the TEN domain, and there is no assigned density for the TEN domain termini implicated in RNA binding. Furthermore, studies have revealed alternative secondary structures for TR regions that could interact with TERT prior to TR folding into active conformation. Informed by recent advances in knowledge of telomerase structure, we returned to the investigation of Tetrahymena thermophila TERT TEN domain interaction with TR. Instead of finding specificity for a particular TR sequence or structure, we discovered that the tagged TEN domain used in previous characterizations has trace contamination with a bacterial RNA-interacting protein not detectable by SDS-PAGE. By resolving this interference, we show that the TEN domain binds RNAs dependent on RNA length rather than sequence.

biochemistry↗