Biology subjects

De Maria, M.

Publications and source records attributed to De Maria, M..

3 recordsLinked to original sources

bioRxiv2024

Retromer Opposes Opioid-Induced Downregulation of the Mu Opioid Receptor

The mu opioid receptor (MOR) is protected from opioid-induced trafficking to lysosomes and proteolytic downregulation by its ability to access the endosomal recycling pathway through its C-terminal recycling motif, LENL. MOR sorting towards the lysosome results in downregulation of opioid signaling while recycling of MOR to the plasma membrane preserves signaling function. However, the mechanisms by which LENL promotes MOR recycling are unknown, and this sequence does not match any known consensus recycling motif. Here we took a functional genomics approach with a comparative genome-wide screen design to identify genes which control opioid receptor expression and downregulation. We identified 146 hits including all three subunits of the endosomal Retromer complex. We show that the LENL motif in MOR is a novel Retromer recycling motif and that LENL is a necessary, sufficient, and conserved mechanism to give MOR access to the Retromer recycling pathway and protect MOR from agonist-induced downregulation to multiple clinically relevant opioids including fentanyl and methadone.

cell biology↗

bioRxiv2023

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification

AbstractThe Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

genomics↗

bioRxiv2022

A genome-wide CRISPR interference screen using an engineered trafficking biosensor reveals a role for RME-8 in opioid receptor regulation

G protein-coupled receptors (GPCRs) are the largest family of membrane-bound signaling molecules. Activity of these receptors is critically regulated by their trafficking through the endo-lysosomal pathway. Identifying the genes involved in GPCR trafficking is challenging due the complexity of sorting operations and low affinity protein-protein interactions. Here we present a chemical biology fluorescence-based technique to interrogate GPCR trafficking. We show that the engineered enzyme APEX2 is a highly sensitive biosensor for GPCR trafficking to the lysosome, and this trafficking can be monitored through APEX-based activation of fluorogenic substrates such as Amplex UltraRed (AUR). We used this approach to perform a genome-wide CRISPR interference screen focused on the delta type opioid receptor (DOR), a GPCR which modulates anxiety, depression, and pain. The screen identified 492 genes including known- and novel-regulators of DOR expression and trafficking. We demonstrate that one of the novel genes, RME-8, localizes to early endosomes and plays a critical role in regulating DOR trafficking to the lysosome. Together, our data demonstrate that GPCR-APEX2/AUR is a flexible and highly sensitive chemical biology platform for genetic interrogation of receptor trafficking.

cell biology↗