Biology subjects

Makhamreh, A.

Publications and source records attributed to Makhamreh, A..

5 recordsLinked to original sources

bioRxiv2025

Direct RNA sequencing of primary human T cells reveals the impact of immortalization on mRNA pseudouridine modifications.

Immortalized cell lines are commonly used as proxies for primary cells in human biology research. For example, Jurkat leukemic T cells fundamentally contributed to uncovering T cell signaling, activation, and immune responses. However, the immortalization process can alter key cellular properties, and researchers widely believe this process could significantly change RNA modification machinery and modification sites. In this study, we focus on pseudouridine ({Psi}), one of the most abundant mRNA modifications, and compare {Psi} profiles in mRNA from primary and immortalized T cells using direct RNA sequencing (DRS). Surprisingly, 87% of {Psi}-sites were shared between the two cell types, primarily in transcripts encoding proteins involved in essential cellular processes, including RNA-modification regulation. Furthermore, the analysis of the 13% of sites unique to each cell type reveals that Jurkat cells contained transcripts linked to immune activation and oncogenesis, while primary T cells contained transcripts associated with calcium signaling and intracellular trafficking. We provide a list of these genes, which should be considered when using immortalized cells to study RNA modifications in immunology contexts. Most differences were driven by whether the mRNA was present or absent in the immortalized or primary cell type. Interestingly, RNA-modification enzyme expression levels were highly conserved in both cell types. This suggests that site-specific differences in {Psi} levels arise from regulatory processes acting in trans rather than differences in modification enzyme levels. Significance StatementIt is widely believed that RNA modification machinery and modification sites could be significantly altered in immortalized cells, yet this has never been tested. Focusing on pseudouridine ({Psi}), we map {Psi} in the transcriptomes of primary and immortalized human T cells. Surprisingly, most sites are conserved in the primary and immortalized T cells, with several important examples of cases with cell type specificity and should be considered on a case-by-case basis. Furthermore, we evaluated RNA-modification machinery levels in primary and immortalized T cells, finding high conservation across the cell lines. Our findings demonstrate that RNA modifications are largely conserved between primary and immortalized cells, and the edge cases can be considered individually.

genomics↗

bioRxiv2024

Probing enzyme-dependent pseudouridylation using direct RNA sequencing to assess neuronal epitranscriptome plasticity

Chemical modifications in mRNAs, such as pseudouridine (psi), can control gene expression. Yet, we know little about how they are regulated, especially in neurons. We applied nanopore direct RNA sequencing to investigate psi dynamics in SH-SY5Y cells in response to two perturbations that model a natural and unnatural cellular state: retinoic-acid-mediated differentiation (healthy) and exposure to the neurotoxicant, lead (unhealthy). We discovered that the expression of some psi writers change significantly in response to physiological conditions. We also found that globally, lead-treated cells have more psi sites but lower relative occupancy than untreated cells and differentiated cells. Interestingly, examples of highly plastic sites were accompanied by constant expression for psi writers, suggesting trans-regulation. Many positions were static throughout all three cellular states, suggestive of a "housekeeping" function. This study enables investigations into mechanisms that control psi modifications in neurons and its possible protective effects in response to cellular stress.

systems biology↗

bioRxiv2022

Messenger-RNA Modification Standards and Machine Learning Models Facilitate Absolute Site-Specific Pseudouridine Quantification

Enzyme-mediated chemical modifications to mRNA are important for fine-tuning gene expression, but they are challenging to quantify due to low copy number and limited tools for accurate detection. Existing studies have typically focused on the identification and impact of adenine modifications on mRNA (m6A and inosine) due to the availability of analytical methods. The pseudouridine ({Psi}) mRNA modification is also highly abundant but difficult to detect and quantify because there is no available antibody, it is mass silent, and maintains canonical basepairing with adenine. Nanopores may be used to directly identify {Psi} sites in RNAs using a systematically miscalled base, however, this approach is not quantitative and highly sequence dependent. In this work, we apply supervised machine learning models that are trained on sequence-specific, synthetic controls to endogenous transcriptome data and achieve the first quantitative {Psi} occupancy measurement in human mRNAs. Our supervised machine learning models reveal that for every site studied, different signal parameters are required to maximize {Psi} classification accuracy. We show that applying our model is critical for quantification, especially in low-abundance mRNAs. Our engine can be used to profile {Psi}-occupancy across cell types and cell states, thus providing critical insights about physiological relevance of {Psi} modification to mRNAs.

genomics↗

bioRxiv2021

Detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct, long-read sequencing

We developed and applied a semi-quantitative method for high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identified many known sites of pseudouridylation and uncovered new uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites were validated using 1,000-mer synthetic RNA controls bearing a single pseudouridine in the center position which demonstrate systematical under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our pipeline allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data as well as multiple modifications on the same strand.

bioengineering↗

bioRxiv2021

Unidirectional Single-File Transport of Full-Length Proteins Through a Nanopore

Nanopore sensors could revolutionize single-molecule proteomics by providing a means for identification of known proteins through fingerprinting or by de novo sequencing. However, the complex chemical and physical properties of proteins present multiple challenges to the conventional nanopore sensing method, predominantly, the single-file threading of a protein chain into a nanopore and its transport through it. Herein we describe a general approach for realizing unidirectional transport of full-length proteins through nanopores. We show that the combination of a chemically resistant biological nanopore platform and a high concentration guanidinium chloride buffer enables protein unfolding and unidirectional transport through a pore, propelled by an electroosmotic effect that largely owes to the guanidinium chloride presence. The uniform and slow (~10 {micro}s/amino acid) single-file transport, when combined with supervised machine learning of the electrical current signatures obtained, allows us to use to discern the protein threading orientation and identity. In conjunction with a method for tail-modification of native proteins and higher-resolution nanopores, our approach could offer a path towards direct single-molecule protein fingerprinting without the requirement of a motor enzyme.

biophysics↗