Biology subjects

Lim, H. M.

Publications and source records attributed to Lim, H. M..

5 recordsLinked to original sources

bioRxiv2024

Rho-dependent termination and RNase E-mediated cleavage: Dual pathways for RNA 3' end processing in polycistronic mRNA

"Pre-full-length" transcripts are produced at the end of the polycistronic galactose (gal) operon, 5galE-galT-galK-galM3, via Rho-dependent (RDT) and -independent transcription termination (RIT). The full-length galETKM mRNAs 3 end is acquired by exo-nucleolytic processing of the 3-OH ends of the pre-full-length transcripts. However, the gal operon produces an mRNA named galE whose 3 end forms at [~]120 nucleotides from the galE stop codon, thus in the following gene, galT, establishing polarity in gene expression. In this study, we investigated the molecular processes that generate the 3 end of galE mRNA. We discovered that the 3 ends of pre-galE mRNA are produced in the middle of the galT gene as a result of the combination of two separate molecular processes - one previously reported as RDT and the other as unreported RNase E-mediated transcript cleavage. The 3 ends of the pre-galE mRNA are exo-nucleolytically processed to the current 3 end of the galE mRNA. A hairpin structure of 8 base-pair stems and 4 nucleotide-loop formed 5-10 nucleotides upstream of the 3 ends of the galE mRNA blocks the exoribonuclease digestion and renders stability. These findings showed that RNase E produces RNA 3end establishing polarity in gene expression, in contrast to the general role of mRNA degradation. Significance statementHere, we show the findings of two molecular mechanisms that generate the pre-galE mRNA 3ends in the gal operon: Rho-dependent termination (RDT) and RNase E-mediated cleavage. These 3 ends are subsequently processed to produce stable galE mRNA with a hairpin structure that prevents exoribonuclease degradation. This mechanism establishes gene expression polarity by generating the 3 end of galE mRNA within the galT gene, contrasting with the usual mRNA degradation role of RNase E. The study reveals a unique role of RNase E in mRNA processing and stability.

microbiology↗

bioRxiv2024

Deciphering the coupling state-dependent transcription termination in the Escherichia coli galactose operon

The distance between the ribosome and the RNA polymerase active center, often referred to as the mRNA loop length, is a critical determinant of transcription-translation coupling. While structural biology studies have indicated the existence of multiple expressomes with varying mRNA loop lengths, their in vivo roles and functional significance remain largely unexplored. This study delves into the mechanisms governing transcription termination within the Escherichia coli galactose operon, revealing a crucial role in the transcription and translation coupling state. The operon employs both Rho-independent and Rho-dependent terminators. Our findings demonstrate that long-loop coupled transcription-translation complexes preferentially terminate at the upstream Rho-independent terminator. In contrast, short-loop coupled complexes bypass the Rho-independent terminator and terminate at the downstream Rho-dependent terminator. The efficiency of the Rho-independent terminator is enhanced by an extended U-track, suggesting a novel mechanism for overcoming ribosome inhibition. These results challenge the traditional view of transcription termination as a random process, highlighting a predetermined mechanism contingent on the coupling state. This study emphasizes the intricate interactions between transcription and translation in prokaryotes. Understanding how these processes affect the RNA polymerases selection of transcriptional terminators is critical for developing strategies to regulate gene expression.

molecular biology↗

bioRxiv2023

Quantification of Rho-termination in vivo using qRT-PCR: a comprehensive analysis

In prokaryotes, the Rho protein mediates Rho-dependent termination (RDT) by identifying a non-specific cytosine-rich Rho utilization site on the newly synthesized RNA. As a result of RDT, downstream RNA transcription is reduced. Due to the bias in reverse transcription and PCR amplification, we were unable to identify the RDT site by directly measuring the amount of mRNA upstream and downstream of RDT sites. To overcome this difficulty, we employed a 77 bp reporter gene argX, coding transfer RNA that binds L-arginine, tRNAarg from Brevibacterium albidum, and transcriptionally fused it to the sequences to be assayed. We constructed a series of plasmids by combining a segment of the galactose (gal) operon sequences, both with and without the RDT regions at the ends of cistrons (galE, galT, and galM) upstream of argX. The RNA polymerase will transcribe the gal operon sequence and argX unless it encounters the RDT encoded by the inserted sequence. We observed similar tRNAarg half-lives expressed in these transcriptional fusion plasmids. Therefore, the amount of tRNAarg directly represents the number of transcripts transcribed. Using this approach, we were able to effectively assay the RDTs in the gal operon by quantifying the relative amount of tRNAarg using quantitative real-time PCR (qRT-PCR) analyses. The resultant RDT% for galET, galTK, and at the end of galM were 36, 26, and 63, individually. Our findings demonstrate that combining tRNAarg, with qRT-PCR can directly measure RDT efficiencies in vivo, making it a useful tool for gene expression research.

synthetic biology↗

bioRxiv2022

Identification of Rho-dependent termination site in vivo using synthetic sRNA

Rho promotes Rho-dependent termination (RDT) at the Rho-dependent terminator, producing a variable-length region at the 3'-end of mRNA without secondary structure. Determining the exact RDT site in vivo is challenging because the 3'-end of mRNA is rapidly removed by 3'- to 5'-exoribonuclease digestion after RDT. Here, we applied synthetic sRNA (sysRNA) to pinpoint RDT sites in vivo by exploiting its complementary base-pairing ability to target mRNA. Through the combined assays of rapid amplification of cDNA 3'-ends, primer extension, and capillary electrophoresis, we could precisely locate and quantify mRNA 3'-ends. We found that complementary double-stranded RNA (dsRNA) formed between sysRNA and mRNA was efficiently cleaved by RNase III in the middle of the dsRNA region. The formation of dsRNA seems to protect the cleaved RNA 3'-ends from rapid degradation by 3'- to 5'-exonuclease, thereby stabilizing the mRNA 3'-end. We further verified that the signal intensity at the 3'-end was positively correlated with amounts of mRNA. By constructing a series of sysRNAs with target sites in close proximity, and comparing the difference in signal intensity at the 3'-end of wild-type and Rho-impaired strains, we finally identified a region of increased mRNA expression within 21 bp range, which was determined as RDT site. Our results demonstrated the ability to use sysRNA as a novel tool to precisely localize RDTs in vivo and expanded the range of sysRNA applications. IMPORTANCEWith the emergence of more new tools for inhibiting gene expression, sysRNA, which was once widely used, has gradually faded out of people's attention due to its unstable inhibition effect and low inhibition efficiency. However, it remains an interesting topic as a regulatory tool due to its ease of design and low metabolic burden on cells. Here, for the first time, we discovered a new function to identify RDT sites in vivo using sysRNA. This new feature is important because, since the discovery of the Rho protein in 1969, it has been difficult to specifically identify RDT sites in vivo due to the rapid processing of RNA 3'-ends by exonucleases, and sysRNA might provide a new way to address this challenge.

synthetic biology↗

bioRxiv2020

AIM2 Inflammasome Mediates Hallmark Neuropathological Alterations and Cognitive Impairment in a Mouse Model of Vascular Dementia

Chronic cerebral hypoperfusion is associated with vascular dementia (VaD). Cerebral hypoperfusion may initiate complex molecular and cellular inflammatory pathways that contribute to long-term cognitive impairment and memory loss. Here we used a bilateral common carotid artery stenosis (BCAS) mouse model of VaD to investigate its effect on the innate immune response - particularly the inflammasome signaling pathway. Comprehensive analyses revealed that chronic cerebral hypoperfusion induces a complex temporal expression and activation of inflammasome components and their downstream products (IL-1{beta} and IL-18) in different brain regions, and promotes activation of apoptotic and pyroptotic cell death pathways. Polarized glial cell activation, white matter lesion formation and hippocampal neuronal loss also occurred in a spatiotemporal manner. Moreover, in AIM2 knockout mice we observed attenuated inflammasome-mediated production of proinflammatory cytokines, apoptosis and pyroptosis, as well as resistance to chronic microglial activation, myelin breakdown, hippocampal neuronal loss, and behavioural and cognitive deficits following BCAS. Hence, we have demonstrated that activation of the AIM2 inflammasome substantially contributes to the pathophysiology of chronic cerebral hypoperfusion-induced brain injury and may therefore represent a promising therapeutic target for attenuating cognitive impairment in VaD.

neuroscience↗