Applying the pro‐siRNA Technology to Understand RNA Transcription and Processing

Abstract

RNA interference (RNAi), a process where messenger RNA is deactivated by small interfering RNAs or microRNAs, has been extensively used in biological research for the study of gene functions in mammalian cells. A type of ~21-nucleotide (nt) siRNAs, termed prokaryotic small interfering RNA (pro-siRNA), was isolated and purified from Escherichia coli cells co-expressing the plant Tombusvirus p19 and sense-antisense sequences of exogenous genes. These pro-siRNAs exhibit excellent knockdown efficiencies on targeting exogenous genes in mammalian cells, while inducing minimal off-target effects. Based on this technology, the first aim of my PhD project is to utilize a transcriptome-wide RNAi screen approach to investigate an important biological process. The second aim is to investigate the functions of RNase III enzymes from various bacterial species, which are endonucleases responsible for processing double-stranded RNAs. The final aim is to apply the pro-siRNA technology to study antisense RNA transcription and processing in Pseudomonas aeruginosa, an important bacterial pathogen. The findings significantly contributed to the understanding of RNA transcription regulation in mammalian cells and the mechanism of RNA processing in bacterial cells.

I have successfully produced a high quality pro-siRNA library for a human cell model. To achieve this, a streamlined protocol for high throughput production of highly potent pro-siRNAs was devised. Several crucial steps including pro-siRNA library plasmid construction, bacterial incubation and pro-siRNA purification were optimized. A customized pro-siRNA library containing 2,496 pro-siRNAs with an average yield of 2±0.5 µg siRNA per well was produced for HeLa-d1EGFP cells in 96-well plates. Transfection experiments showed that most pro-siRNAs selected from the library were highly potent and able to silence target genes by more than 80% in the HeLa cells. Using the library an RNAi screen was performed, which identified ATF7IP, UBA2, HNRNPU, HNRNPA1 and SPTBN1 as regulators inhibiting stable transgene expressions, driven by the CMV promoter, in the HeLa-d1EGFP reporter cell line. RNAi or CRISPR-Cas9 mediated inhibitions of ATF7IP, a transcription factor recruiter and heterochromatin formation regulator, increased the expressions of recombinant monoclonal antibody in HeLa and CHO-K1 cells, in which the effects were magnified by co-inhibitions of UBA2 or HNRNPU. These data suggested that ATF7IP, UBA2 and HNRNPU are involved in the regulation of CMV promoter-driven transcription in mammalian cells.

The production of pro-siRNAs is mediated by RNase III in bacterial cells. The specificity of RNase III could affect the efficiency and target sequence distribution of pro-siRNAs. Ectopic expressions of various RNase III homologs in an RNase III mutant E. coli strain HT115 (DE3) has not only enabled the investigation on RNase III functional diversity among different bacterial species, but also empowered the productions of diverse pro-siRNAs. In addition, the pro-siRNA plasmid can be modified based on the knowledge on RNase III sequence specificity to improve RNAi efficiency. The findings revealed novel insights into the molecular mechanisms of RNase III in bacteria and resulted in the development of new tools for pro-siRNA bioengineering.

Endogenous antisense RNAs (asRNAs) overlap with complementary sense RNAs to form double stranded RNAs (dsRNAs), which are cleaved by RNase III in bacteria. Ectopic expression of p19 in Pseudomonas aeruginosa allowed us to purify the highly unstable ~21 nt RNase III cleavage intermediates for characterising endogenous dsRNAs in the bacteria by deep sequencing. The results demonstrated that it is a sensitive method to investigate the distributions of antisense transcripts across the genomic loci and RNase III cleavage patterns of dsRNA formed by overlapping sense and antisense transcripts in Pseudomonas aeruginosa.

In conclusion, the pro-siRNA technology allowed us to produce a pro-siRNA library for transcriptome-wide RNAi screen for functional genomics in HeLa-d1EGFP cells, which could potentially be customized for RNAi screens in any other eukaryotic cells. It has also enabled us to study vital biological processes including but not limited to RNase III-initiated double-stranded RNA degradation and antisense RNA transcription, which could contribute significantly to the study of gene regulation in bacteria.

Date of Award	27 Jul 2022
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Xin DENG (Supervisor) & Linfeng HUANG (External Co-Supervisor)