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Keegan, L. P.

Publications and source records attributed to Keegan, L. P..

5 recordsLinked to original sources
bioRxiv2024

Global Analysis by LC-MS/MS of N6-Methyladenosine and Inosine in mRNA Reveals Complex Incidence

The precise and unambiguous detection and quantification of internal RNA modifications represents a critical step for understanding their physiological functions. The methods of direct RNA sequencing are quickly developing allowing for the precise location of internal RNA marks. This detection is however not quantitative and still presents detection limits. One of the biggest remaining challenges in the field is still the detection and quantification of m6A, m6Am and m1A modifications. The second intriguing and timely question remaining to be addressed is the extent to which individual marks are coregulated or potentially can affect each other. Here we present a methodological approach to detect and quantify several key mRNA modifications in human total RNA and in mRNA, which is difficult to purify way from contaminating tRNA. We show that the adenosine demethylase FTO primarily targets m6Am marks in noncoding RNAs in HEK293T cells. Surprisingly, we observe little effect of FTO or ALKBH5 depletion on the m6A mRNA levels. Interestingly, upregulation of ALKBH5 is accompanied by an increase in inosine level in overall mRNA.

molecular biology↗
bioRxiv2023

Amyloid precursor protein induces reactive astrogliosis

We present in vitro and in vivo evidence demonstrating that Amyloid Precursor Protein (APP) acts as an essential instigator of reactive astrogliosis. Cell-specific overexpression of APP in cultured astrocytes led to remodelling of the intermediate filament network, enhancement of cytokine production and activation of cellular programs centred around the interferon (IFN) pathway, all signs of reactive astrogliosis. Conversely, APP deletion in cultured astrocytes abrogated remodelling of the intermediate filament network and blunted expression of IFN stimulated gene (ISG) products in response to lipopolysaccharide (LPS). Following traumatic brain injury (TBI), mouse reactive astrocytes also exhibited an association between APP and IFN, while APP deletion curbed the increase in glial fibrillary acidic protein (GFAP) observed canonically in astrocytes in response to TBI. Thus, APP represents a molecular inducer and regulator of reactive astrogliosis.

neuroscience↗
bioRxiv2023

Aberrant activation of the innate immune sensor PKR by self dsRNA is prevented by direct interaction with ADAR1

Loss of dsRNA editing by Adar1 leads to aberrant interferon induction in Adar null mouse embryos. Adar Mavs mutants, in which this interferon induction is prevented, die within two weeks of birth. We show here that early death of pups is reduced in Adar Mavs EIF2k2 (Pkr) mutants, identifying PKR as the second aberrantly activated dsRNA sensor in Adar mutant mice. In intestines of Adar Mavs pups transit amplifying progenitor cells in intestinal crypts die and intestinal villi are lost in pups before death; intestinal defects are prevented in Adar Mavs Eifak2. In human A549 lung cancer cells ADAR1 forms a complex with PKR. AlphaFold modelling predicts a direct inhibitory interaction of ADAR1 dsRBDIII with the PKR near the kinase active site and a new mode for ADAR1 dsRNA-binding. Mutations at residues required for ADAR1 dsRNA binding or for predicted PKR contacts, prevent interaction with PKR.

immunology↗
bioRxiv2019

The evolution of the 9aaTAD domain in Sp2 proteins: inactivation with valines and intron reservoirs

The Sp1 transcription factor has been defined as glutamine-rich activator. The Nine amino acid TransActivation Domains (9aaTAD) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in the Sp1 and in all nine members of SP family with broad natural 9aaTAD variations. We showed by the amino acid substitutions that the glutamine residues are completely dispensable for 9aaTADs function. We described the 9aaTAD domains origin and evolutionary history. The ancestral Sp2 gene with inactive 9aaTAD has duplicated in early chordates and created new paralogs Sp1, Sp3 and Sp4. We discovered that the accumulation of valines in the 9aaTADs correlated with the domain inactivation. The Sp2 activation domain, whose dormancy have lasted over 100 million years during chordate evolution, enabled later diversification in the Sp1-4 clade, including both repressors and activators. The new paralogs Sp1 and Sp3 activation domains have regained their original activator function by loss of valines in their 9aaTADs.

molecular biology↗