Biology subjects

Mirza, A. H.

Publications and source records attributed to Mirza, A. H..

6 recordsLinked to original sources

bioRxiv2022

Selective detection of m6A derived from mRNA using the Phospho-tag m6A assay

N6-methyladenosine (m6A) is a modified nucleotide found in mRNA, ribosome RNA (rRNA) and small nuclear RNA (snRNA). m6A in mRNA has important roles in regulating mRNA stability, splicing, and other processes. Numerous studies have described m6A as a dynamic modification using mass spectrometry-based quantification of m6A in mRNA samples prepared from different cellular conditions. However, these results have been questioned based on the finding that the mRNA purification protocols often result in varying levels of rRNA contamination. Additionally, mRNA purification protocols disproportionately enrich for the 3 ends of mRNA, a region that is enriched in m6A. To address these problems, we developed the Phospho-tag m6A assay, a highly efficient method for quantifying m6A specifically from mRNA. In this assay, a series of selective RNase digestion steps is performed, which results in m6A from rRNA and snRNA being liberated as m6A monophosphate, while m6A from mRNA is mostly liberated as m6A nucleoside. m6A levels are normalized to transcript levels, using m7G monophosphate liberated by yDcpS decapping enzyme as a surrogate for mRNA levels. Notably, this approach uses total cellular RNA, rather than purified mRNA, which simplifies the steps for m6A detection and overcomes the 3-end biases associated with mRNA purification. Overall, the Phospho-tag m6A provides a simple and efficient method for quantification of mRNA-derived m6A from total RNA samples.

molecular biology↗

bioRxiv2021

Understanding the source of METTL3-independent m6A in mRNA

N6-methyladenosine (m6A) is a highly prevalent mRNA modification which promotes degradation of transcripts encoding proteins that have roles in cell development, differentiation, and other pathways. METTL3 is the major methyltransferase that catalyzes the formation of m6A in mRNA. As 30-80% of m6A can remain in mRNA after METTL3 depletion by CRISPR/Cas9-based methods, other enzymes are thought to catalyze a sizable fraction of m6A. Here, we re-examined the source of m6A in the mRNA transcriptome. We characterized mouse embryonic stem cell lines which continue to have m6A in their mRNA after Mettl3 knockout. We show that these cells express alternatively spliced Mettl3 transcript isoforms that bypass the CRISPR/Cas9 mutations and produce functionally active methyltransferases. We similarly show that other reported METTL3 knockout cell lines express altered METTL3 proteins. We find that gene dependency datasets show that most cell lines fail to proliferate after METTL3 deletion, suggesting that reported METTL3 knockout cell lines express altered METTL3 proteins rather than have full knockout. Finally, we reassessed METTL3s role in synthesizing m6A using a genomic deletion of Mettl3, and found that METTL3 is responsible for >95% of m6A in mRNA. Overall, these studies suggest that METTL3 is responsible for the vast majority of m6A in the transcriptome, and that remaining m6A in putative METTL3 knockout cell lines is due to the expression of altered but functional METTL3 isoforms.

molecular biology↗

bioRxiv2020

Transcriptome profiling of ulcerative colitis mouse model suggests biomarkers and therapeutic targets for human colitis

1.BACKGROUND & AIMSUlcerative colitis (UC) is an inflammatory bowel disorder with unknown etiology. Given its complex nature, in vivo studies to investigate its pathophysiology is vital. Animal models play an important role in molecular profiling necessary to pinpoint mechanisms that contribute to human disease. Thus, we aim to identify common conserved gene expression signatures and differentially regulated pathways between human UC and a mouse model hereof, which can be used to identify UC patients from healthy individuals and to suggest novel treatment targets and biomarker candidates. METHODSTherefore, we performed high-throughput total and small RNA sequencing to comprehensively characterize the transcriptome landscape of the most widely used UC mouse model, the dextran sodium sulfate (DSS) model. We used this data in conjunction with publicly available human UC transcriptome data to compare gene expression profiles and pathways. RESULTSWe identified differentially regulated protein-coding genes, long non-coding RNAs and microRNAs from colon and blood of UC mice and further characterized the involved pathways and biological processes through which these genes may contribute to disease development and progression. By integrating human and mouse UC datasets, we suggest a set of 51 differentially regulated genes in UC colon and blood that may improve molecular phenotyping, aid in treatment decisions, drug discovery and the design of clinical trials. CONCLUSIONGlobal transcriptome analysis of the DSS-UC mouse model supports its use as an efficient high-throughput tool to discover new targets for therapeutic and diagnostic applications in human UC through identifying relationships between gene expression and disease phenotype.

molecular biology↗

bioRxiv2020

Identification of a Micropeptide on Lipid Droplet That Mediates Lipid Storage and Insulin Sensitivity

Over the past decade, great progress in sequencing technologies and computational biology has revealed that the majority of the mammalian genome considered to be noncoding is rich in functional elements able to produce proteins. Many RNA molecules, mis-annotated as noncoding, actually harbor small open reading frames that are predicted to code for proteins. Some of those proteins have been verified to play critical roles in multiple biological processes. The lipid droplet (LD) is a unique cellular organelle, conserved from bacteria to humans, and is closely associated with cellular lipid metabolism and metabolic disorders. No noncoding RNA-coded proteins have been identified on LDs. Here, for the first time, we searched the organelle for their presence. After the enrichment of small proteins of LDs isolated from myoblasts, we used mass spectrometry coupled with our lab made protein database to identify LD-associated noncoding RNA-encoded proteins (LDANPs). A total of 15 new proteins were identified. One of them was studied further and termed LDANP1. LDANP1 was localized on LDs by imaging, cell fractionation, and immunogold labeling. Like LD resident proteins, LDANP1 was degraded by the proteasome. Using the CRISPR/Cas9-mediated genome editing technique, the endogenous expression of LDANP1 was validated. The stable expression of LDANP1 suppressed the accumulation of triacylglycerol in oleic acid treated myoblasts and inhibited the rescue of palmitate-inhibited insulin sensitivity by oleic acid. In summary, we report for the first time that translatable, nominally noncoding RNA-derived proteins, which are new and cannot be identified using current research methods, were associated with LDs and that among these, LDANP1 modulated lipid metabolism and insulin sensitivity. The discovery of noncoding RNA-encoded proteins on LDs paves a new way for the research of LDs and lipid metabolism.

cell biology↗

bioRxiv2020

Lipid Droplet-Anchored Mitochondria Are More Sensitive to Cold in Brown Adipocytes

Brown adipose tissue (BAT) are specialized for uncoupled heat production through mitochondria fueled majorly from fatty acids (FA) of lipid droplets (LDs). How the interaction between the two organelles contributes the generation of heat remains elusive. Here we report that LD-anchored mitochondria (LDAM) were observed in BAT of mice raised at three different temperatures, 30{degrees}C, 23{degrees}C, and 6{degrees}C. The biochemical analyses including Western blotting and electron transport chain subunits showed that LDAM were functional at given temperatures. Comparative proteomics analysis was conducted and revealed that these LDAM had protein level differences from cytoplasmic mitochondria (CM) at different temperatures. Higher expressions of proteins at low temperature were observed for i) FA {beta}-oxidation in LDAM including FA synthesis, and uncoupling, ii) pseudo-futile cycle in CM, and iii) two shuttle systems; glycerol 3-phosphate in both CM and LDAM, and citrate malate in CM. Together, these results suggest that LDs and LDAM are a preorganized and functional organelle complex that permits the rapid response to cold environment.

cell biology↗

bioRxiv2020

Atomistic Molecular Dynamics Simulations of Trioleoylglycerol - Phospholipid Membrane Systems

Adiposomes are phospholipid coated triacylglyceride particles that serve as structural models of the fat storage compartments of cells, known as lipid droplets (LDs); however, unlike LDs, they do not carry proteins. There is a deficit of available methods and experimental data regarding the internal packing of the adiposomes, and computer simulations offer a promising way to pinpoint the molecular arrangements within these structures. However, in the absence of a triacylglycerol-specific atomic forcefield, thus far, all adiposome/LD simulations have been performed with the coarse grained/united atom forcefields. Yet it is desirable to model the phospholipid/triacylglycerol interface with atomic resolution. In the present study, we first prepared a 2-monooleoylglycerol (MOG) forcefield which was then used to build a trioleoylglycerol (TOG) forcefield by the modular approach of the AMBER software suite. TOG bilayer membrane (2L) systems were modelled from two different initial conformations; TOG3 and TOG2:1. The simulations revealed that TOG2:1 is the most populated conformation in TOG membranes, irrespective of the starting conformation. Some other parameter optimizations were performed for TOG membranes based on which adiposome mimicking tetralayer membrane system (4L) was prepared with a TOG bilayer at core surrounded by two DOPC leaflets. The 4L membranes were stable throughout the simulations, however it was observed that a small amount of cations and water diffused from surface to the TOG core of the membrane. Based on these results a TAG-packing model was also developed. It is expected that the availability of MOG forcefield will equip future studies with a framework for molecular dynamics simulations of adiposomes/LDs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/918136v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@e79991org.highwire.dtl.DTLVardef@113291corg.highwire.dtl.DTLVardef@f02014org.highwire.dtl.DTLVardef@ca4227_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗