Biology subjects

Catrina, I. E.

Publications and source records attributed to Catrina, I. E..

4 recordsLinked to original sources

bioRxiv2023

TFOFinder: Python program for identifying purine-only double-stranded stretches in the predicted secondary structure(s) of RNA targets

Nucleic acid probes are valuable tools in biology and chemistry and are indispensable for PCR amplification of DNA, RNA quantification and visualization, and downregulation of gene expression. Recently, triplex forming oligonucleotides (TFO) have received increased attention due to their improved selectivity and sensitivity in recognizing purine-rich double-stranded RNA regions at physiological pH by incorporating backbone and base modifications. For example, triplex forming peptide nucleic acid (PNA) oligomers have been used for imaging structured RNA in cells and inhibiting influenza A replication. Although a handful of programs are available to identify triplex target sites (TTS) in DNA, none are available that find such regions in structured RNAs. Here, we describe TFOFinder, a Python program that facilitates the identification of intramolecular purine-only RNA duplexes that are amenable to forming parallel triple helices (pyrimidine/purine/pyrimidine). We performed genome- and transcriptome-wide analyses of TTS in Drosophila melanogaster and found that only 0.3% (123) of total unique transcripts (35,642) show the potential of forming 12-purine long triplex forming sites that contain at least one guanine. Using minimization algorithms, we predicted the secondary structure(s) of these transcripts, and using TFOFinder, we found that 97 (79%) of the identified 123 transcripts are predicted to fold to form at least one TTS for parallel triple helix formation. The number of transcripts with potential purine TTS increases when the strict search conditions are relaxed by decreasing the length of the probes or by allowing up to two pyrimidine inversions or 1-nucleotide bulge in the target site. These results are encouraging for the use of modified triplex forming probes for live imaging of endogenous structured RNA targets, such as pre-miRNAs, and inhibition of target-specific translation and viral replication.

biochemistry↗

bioRxiv2023

Cup is essential for oskar mRNA translational repression during early Drosophila oogenesis

The proper timing of mRNA translation is crucial across many biological systems for processes such as intercellular communication, body pattern formation, and morphogenesis. The main D. melanogaster posterior determinant, oskar, is maternally transcribed, but only translated when properly localized at the oocytes posterior cortex. Bruno 1 and Cup are two effector proteins known to participate in multiple aspects of oskar mRNA regulation. Current model describes a mechanism in which Bruno 1 is necessary for Cups recruitment to oskar mRNA, and Bruno 1 is indispensable for its translational repression. Here, we reveal that the Bruno 1-Cup interaction, as well as their interdependent influence on each others mRNA and protein expression, lead to precise oskar mRNA regulation during early oogenesis. We show that these factors stably associate with the oskar mRNA in vivo, but surprisingly, Bruno 1s stable association with oskar mRNA depends on Cup, while Bruno 1 is not necessary for Cup association to oskar mRNA. During early oogenesis, Cup, not Bruno 1, is the essential factor for oskar mRNA repression. Cup is a crucial P-body member that maintains proper P-body morphology during oogenesis, as well as it is necessary for oskar mRNAs association with P-bodies, thus driving the translational repression and stability of oskar mRNA. Our experimental results collectively suggest a regulatory mechanism where a feedback loop between Bruno 1 and Cup coordinates oskar mRNA regulation in the egg chamber allowing for proper development to occur.

developmental biology↗

bioRxiv2018

PinMol: Python application for designing molecular beacons for live cell imaging of endogenous mRNAs

Molecular beacons are nucleic acid oligomers labeled with a fluorophore and a quencher that fold in a hairpin-shaped structure, which fluoresce only when bound to their target RNA. They are used for the visualization of endogenous mRNAs in live cells. Here, we report a Python program (PinMol) that designs molecular beacons best suited for live cell imaging by using structural information from secondary structures of the target RNA, predicted via energy minimization approaches. PinMol takes into account the accessibility of the targeted regions, as well as the inter- and intramolecular interactions of each selected probe. To demonstrate its applicability, we synthesized an oskar mRNA-specific molecular beacon (osk1236), which is selected by PinMol to target a more accessible region than a manually designed oskar-specific molecular beacon (osk2216). We previously demonstrated osk2216 to be efficient in detecting oskar mRNA in in vivo experiments. Here, we show that osk1236 outperformed osk2216 in live cell imaging experiments.

molecular biology↗

bioRxiv2018

P-bodies and the miRNA pathway regulate translational repression of bicoid mRNA during Drosophila melanogaster oogenesis

Embryonic axis patterning in Drosophila melanogaster is partly achieved by mRNAs that are maternally localized to the oocyte; the spatio-temporal regulation of these transcripts stability and translation is a characteristic feature of oogenesis. While protein regulatory factors are necessary for the translational regulation of some maternal transcripts (e.g. oskar and gurken), small RNA pathways are also known to regulate mRNA stability and translation in eukaryotes. MicroRNAs (miRNAs) are small RNA regulators of gene expression, widely conserved throughout eukaryotic genomes and essential for animal development. The main D. melanogaster anterior determinant, bicoid, is maternally transcribed, but it is not translated until early embryogenesis. We investigated the possibility that its translational repression during oogenesis is mediated by miRNA activity. We found that the bicoid 3UTR contains a highly conserved, predicted binding site for miR-305. Our studies reveal that miR-305 regulates the translation of a reporter gene containing the bicoid 3UTR in cell culture, and that miR-305 only partially contributes to bicoid mRNA translational repression during oogenesis. We also found that Processing bodies (P-bodies) in the egg chamber may play a role in stabilizing bicoid and other maternal transcripts. Here, we offer insights into the possible role of P-bodies and the miRNA pathway in the translational repression of bicoid mRNA during oogenesis.

cell biology↗