Biology subjects

Chin, J.

Publications and source records attributed to Chin, J..

23 records · Page 2Linked to original sources

bioRxiv2021

RBBP6 activates the pre-mRNA 3'-end processing machinery in humans

3-end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3-end of the mature transcript, which is important for mRNA localization, translation and stability. Cleavage must therefore be tightly regulated. Here, we reconstitute specific and efficient 3-endonuclease activity of human CPSF with purified proteins. This requires the sevensubunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm) and, importantly, the multi-domain protein RBBP6. Unlike its yeast homologue Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to human. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in RNA processing.

molecular biology↗

bioRxiv2020

HIV-1 gp120 impairs spatial memory through CREB

HIV-associated neurocognitive disorders (HAND) remains an unsolved problem in the clinical management of HIV-1 carriers, because existing anti-retroviral therapy while suppressing viral replication, do not prevent neurocognitive impairment (e.g. spatial memory loss). HIV-1 gp120 protein has been proposed to contribute to HAND because it is shed by infected cells and the use of antibodies revealed its presence in cerebrospinal fluid (CSF) even in the combinatory antiretroviral therapy (cART) era. The cyclic AMP response element-binding protein (CREB) has long been known to be a star player in memory. CREB exerts its effect partially through regulating the genes for peroxisome proliferator-activated receptor gamma coactivator (PGC)-1 and brain-derived neurotrophic factor (BDNF). CREB, PGC-1, and BDNF levels are low in the brains of patients with neurodegenerative diseases and a dearth of either protein is associated with cognitive decline. We have obtained data showing that gp120 contributes to neurodegeneration by altering CREB phosphorylation on serine residue 133 thus disrupting mitochondrial movement and synaptic plasticity leading to spatial memory loss. Inhibition of CREB function was also associated with a decrease of ATP levels and lower mitochondrial DNA copy numbers. Our data was validated in vitro (primary mouse neurons and neuronal cell line, SH-SY5Y) and in vivo (gp120-tg mice and mice injected with gp120). The negative effect of gp120 was alleviated in cells and animals in the presence of Rolipram. Hence, we conclude that HIV-1 gp120 protein contributes to spatial memory impairment via inhibition of CREB protein activity.

neuroscience↗

bioRxiv2020

TRPM4 conductances in thalamic reticular nucleus neurons generate persistent firing during slow oscillations

During sleep, neurons in the thalamic reticular nucleus (TRN) participate in distinct types of oscillatory activity. While the reciprocal synaptic circuits between TRN and sensory relay nuclei are known to underlie the generation of sleep spindles, the mechanisms regulating slow (<1 Hz) forms of thalamic oscillations are not well understood. Under in vitro conditions, TRN neurons can generate slow oscillations in a cell-intrinsic manner, with postsynaptic Group 1 metabotropic glutamate receptor (mGluR) activation leading to the generation of plateau potentials mediated by both T-type Ca2+ currents and Ca2+ -activated nonselective cation currents (ICAN). However, the identity of ICAN and the possible contribution of thalamic circuits to slow rhythmic activity remain unclear. Using thalamic slices derived from adult mice of either sex, we recorded slow forms of rhythmic activity in TRN neurons, which were mediated by fast glutamatergic thalamoreticular inputs but did not require postsynaptic mGluR activation. For a significant fraction of TRN neurons, synaptic inputs or brief depolarizing current steps led to long-lasting plateau potentials and persistent firing (PF), and in turn, resulted in sustained synaptic inhibition in postsynaptic relay neurons of the ventrobasal thalamus (VB). Pharmacological approaches indicated that plateau potentials were triggered by Ca2+ influx through T-type Ca2+ channels and mediated by Ca2+ and voltage-dependent transient receptor potential melastatin 4 (TRPM4) channels. Taken together, our results suggest that thalamic circuits can generate slow oscillatory activity, mediated by an interplay of TRN-VB synaptic circuits that generate rhythmicity and TRN cell-intrinsic mechanisms that control PF and oscillation frequency. Significance StatementSlow forms of thalamocortical rhythmic activity are thought to be essential for memory consolidation during sleep and the efficient removal of potentially toxic metabolites. In vivo, thalamic slow oscillations are regulated by strong bidirectional synaptic pathways linking neocortex and thalamus. Therefore, in vitro studies in the isolated thalamus can offer important insights about the ability of individual neurons and local circuits to generate different forms of rhythmic activity. We found that circuits formed by GABAergic neurons in the thalamic reticular nucleus (TRN) and glutamatergic relay neurons in the ventrobasal thalamus generated slow oscillatory activity, which was accompanied by persistent firing in TRN neurons. Our results identify both cell-intrinsic and synaptic mechanisms that mediate slow forms of rhythmic activity in thalamic circuits.

neuroscience↗

bioRxiv2019

The potential for polyphosphate metabolism in Archaea and anaerobic polyphosphate formation in Methanosarcina mazei.

Inorganic polyphosphate (polyP) is ubiquitous across all forms of life, but the study of its metabolism has been mainly confined to bacteria and yeasts. Few reports detail the presence and accumulation of polyP in Archaea, and little information is available on its functions and regulation. Here, we report that homologs of bacterial polyP metabolism proteins are present across the major taxa in the Archaea, suggesting that archaeal populations may have a greater contribution to global phosphorus cycling than has previously been recognised. We also demonstrate that polyP accumulation can be induced under strictly anaerobic conditions, in response to changes in phosphate (Pi) availability, i.e. Pi starvation, followed by incubation in Pi replete media (overplus), in cells of the methanogenic archaeon Methanosarcina mazei. Pi-starved M. mazei cells increased transcript abundance of the PHO-regulated alkaline phosphatase (phoA) gene and of the high-affinity phosphate transport (pstSCAB-phoU) operon: no increase in polyphosphate kinase 1 (ppk1) transcript abundance was observed. Subsequent incubation of Pi-starved M. mazei cells under Pi replete conditions, led to a 237% increase in intracellular polyphosphate content and a >5.7-fold increase in ppk1 gene transcripts. Ppk1 expression in M. mazei thus appears not to be under classical PHO regulon control.

microbiology↗

bioRxiv2017

De Novo PacBio long-read and phased avian genome assemblies correct and add to genes important in neuroscience research

Reference quality genomes are expected to provide a resource for studying gene structure and function. However, often genes of interest are not completely or accurately assembled, leading to unknown errors in analyses or additional cloning efforts for the correct sequences. A promising solution to this problem is long-read sequencing. Here we tested PacBio-based long-read sequencing and diploid assembly for potential improvements to the Sanger-based intermediate-read zebra finch reference and Illumina-based short-read Annas hummingbird reference, two vocal learning avian species widely studied in neuroscience and genomics. With DNA of the same individuals used to generate the reference genomes, we generated diploid assemblies with the FALCON-Unzip assembler, resulting in contigs with no gaps in the megabase range (N50s of 5.4 and 7.7 Mb, respectively), and representing 150-fold and 200-fold improvements over the current zebra finch and hummingbird references, respectively. These long-read assemblies corrected and resolved what we discovered to be misassemblies, including due to erroneous sequences flanking gaps, complex repeat structure errors in the references, base call errors in difficult to sequence regions, and inaccurate resolution of allelic differences between the two haplotypes. We analyzed protein-coding genes widely studied in neuroscience and specialized in vocal learning species, and found numerous assembly and sequence errors in the reference genes that the PacBio-based assemblies resolved completely, validated by single long genomic reads and transcriptome reads. These findings demonstrate, for the first time in non-human vocal learning species, the impact of higher quality, phased and gap-less assemblies for understanding gene structure and function.

genomics↗