Fluorescent RNA labeling using self-alkylating ribozymes.

The ability to fluorescently label specific RNA sequences is of significant utility for both in vitro and live cell applications. Currently, most RNA labeling methods utilize RNA-nucleic acid or RNA-protein molecular recognition. However, in the search for improved RNA labeling methods, harnessing the small-molecule recognition capabilities of RNA is rapidly emerging as a promising alternative. Along these lines, we propose a novel strategy in which a ribozyme acts to promote self-alkylation with a fluorophore, providing a robust, covalent linkage between the RNA and the fluorophore. Here we describe the selection and characterization of ribozymes that promote self-labeling with fluorescein iodoacetamide (FIA). Kinetic studies reveal a second-order rate constant that is on par with those of other reactions used for biomolecular labeling. Additionally, we demonstrate that labeling is specific to the ribozyme sequences, as FIA does not react nonspecifically with RNA.

Regulation of sumo mRNA during endoplasmic reticulum stress.

The unfolded protein response (UPR) is a collection of pathways that maintains the protein secretory pathway during the many physiological and pathological conditions that cause stress in the endoplasmic reticulum (ER). The UPR is mediated in part by Ire1, an ER transmembrane kinase and endoribonuclease that is activated when misfolded proteins accumulate in the ER. Ire1's nuclease initiates the cytosolic splicing of the mRNA encoding X-box binding protein (Xbp1), a potent transcription factor that then upregulates genes responsible for restoring ER function. This same nuclease is responsible for the degradation of many other mRNAs that are localized to the ER, through Regulated Ire1 Dependent Decay (RIDD). Here we show that Smt3, a homolog of small ubiquitin-like modifier (sumo), is a non-canonical RIDD target in Drosophila S2 cells. Unlike other RIDD targets, the sumo transcript does not stably associate with the ER membrane, but instead relies on an Xbp1-like stem loop and a second UPR mediator, Perk, for its degradation during stress.

Functional analysis of the microtubule-interacting transcriptome.

RNA localization is an important mechanism for achieving precise control of posttranscriptional gene expression. Previously, we demonstrated that a subset of cellular mRNAs copurify with mitotic microtubules in egg extracts of Xenopus laevis. Due to limited genomic sequence information available for X. laevis, we used RNA-seq to comprehensively identify the microtubule-interacting transcriptome of the related frog Xenopus tropicalis. We identified ~450 mRNAs that showed significant enrichment on microtubules (MT-RNAs). In addition, we demonstrated that the MT-RNAs incenp, xrhamm, and tpx2 associate with spindle microtubules in vivo. MT-RNAs are enriched with transcripts associated with cell division, spindle formation, and chromosome function, demonstrating an overrepresentation of genes involved in mitotic regulation. To test whether uncharacterized MT-RNAs have a functional role in mitosis, we performed RNA interference and discovered that several MT-RNAs are required for normal spindle pole organization and γ-tubulin distribution. Together, these data demonstrate that microtubule association is one mechanism for compartmentalizing functionally related mRNAs within the nucleocytoplasmic space of mitotic cells and suggest that MT-RNAs are likely to contribute to spindle-localized mitotic translation.