Here, we describe a protocol to assess RNA-RNA interactions in situ using an adapted proximity ligation assay (PLA). We detail steps to perform RNA-probe hybridization, in situ rolling circle amplification, and immunofluorescence confocal microscopy. With these tools, it is possible to detect and characterize the intracellular localization of interacting RNA pairs using small cell numbers. This protocol provides a targeted approach to understanding RNA-RNA interactions in intact cells that can complement other established deep-sequencing-based approaches. For complete details on the use and execution of this protocol, please refer to Basavappa et al. (2022)..
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http://dx.doi.org/10.1016/j.xpro.2022.101892 | DOI Listing |
Plants (Basel)
December 2024
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, Russia.
Among the long non-coding RNAs that are currently recognized as important regulatory molecules influencing a plethora of processes in eukaryotic cells, circular RNAs (circRNAs) represent a distinct class of RNAs that are predominantly produced by back-splicing of pre-mRNA. The most studied regulatory mechanisms involving circRNAs are acting as miRNA sponges, forming R-loops with genomic DNA, and encoding functional proteins. In addition to circRNAs generated by back-splicing, two types of circRNAs capable of autonomous RNA-RNA replication and systemic transport have been described in plants: viroids, which are infectious RNAs that cause a number of plant diseases, and retrozymes, which are transcripts of retrotransposon genomic loci that are capable of circularization due to ribozymes.
View Article and Find Full Text PDFCells must limit RNA-RNA interactions to avoid irreversible RNA entanglement. Cells may prevent deleterious RNA-RNA interactions by genome organization to avoid complementarity however, RNA viruses generate long, perfectly complementary antisense RNA during replication. How do viral RNAs avoid irreversible entanglement? One possibility is RNA sequestration into biomolecular condensates.
View Article and Find Full Text PDFWiley Interdiscip Rev RNA
January 2025
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
Coronaviruses utilize a positive-sense single-strand RNA, functioning simultaneously as mRNA and the genome. An RNA-dependent RNA polymerase (RdRP) plays a dual role in transcribing genes and replicating the genome, making RdRP a critical target in therapies against coronaviruses. This review explores recent advancements in understanding the coronavirus transcription machinery, discusses it within virus infection context, and incorporates kinetic considerations on RdRP activity.
View Article and Find Full Text PDFIran J Biotechnol
July 2024
Thoracic surgery Department, Tianjin Chest hospital, Tianjin City, 300222, PR. China.
Background: Long non-coding RNA (lncRNA) U731166 and microRNA (miR)-3607-3p are two ncRNAs with critical roles in cancer biology, while their involvement in esophageal squamous-cell carcinomas (ESCC) is unclear. We predicted that U731166 and miR-3607-3p might interact with each other. This study aimed to investigate their role and interaction in ESCC.
View Article and Find Full Text PDFMol Cell
December 2024
Biotechnology Center, Center for Molecular and Cellular Bioengineering, TU Dresden, Dresden 01307 Saxony, Germany; Cluster of Excellence Physics of Life, TU Dresden, Dresden 01307 Saxony, Germany. Electronic address:
Ribonucleoprotein (RNP) granules have been linked to translation regulation and disease, but their assembly and regulatory mechanisms are not well understood. Here, we show that the RNA-binding protein G3BP1 preferentially interacts with unfolded RNA, driving the assembly of RNP granule-like condensates that establish RNA-RNA interactions. These RNA-RNA interactions limit the mobility and translatability of sequestered mRNAs and stabilize the condensates.
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