Owczarek, E. P. | Search bioRxiv

bioRxiv2020

High-throughput siRNA screening reveals functional interactions and redundancies of human processive exoribonucleases

Processive exoribonucleases, the executors of RNA decay, participate in multiple physical and functional interactions. Unlike physical ones, functional relationships have not been investigated in human cells. Here we have screened cells deficient in DIS3, XRN2, EXOSC10, DIS3L, and DIS3L2 with a custom siRNA library and determined their functional interactions with diverse pathways of RNA metabolism. We uncover a complex network of positive interactions that buffer alterations in RNA degradation. We reveal important reciprocal actions between RNA decay and transcription and explore alleviating interactions between RNA splicing and DIS3 mediated degradation. We also use a large scale library of genes associated with RNA metabolism to determine genetic interactions of nuclear DIS3 and cytoplasmic DIS3L, revealing their unique functions in RNA degradation and uncovering cooperation between the cytoplasmic degradation and nuclear processing of RNA. Finally, genome-wide siRNA screening of DIS3 reveals processes such as microtubule organization and regulation of telomerase activity that are also functionally associated with nuclear exosome-mediated RNA degradation.

systems biology↗

bioRxiv2017

Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. It may also be time-consuming if a homozygote is needed. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternative. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and creation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. The collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the utility of commonly used inducers of tetracycline-responsive promoters. We determined the protein interaction network of the exoribonuclease XRN2 and examined the role of the protein in transcription termination by RNAseq analysis of cells devoid of its ribonucleolytic activity. In total we created more than 500 DNA constructs which proves high efficiency of our strategy.

molecular biology↗