Biology subjects

Correa, I. R.

Publications and source records attributed to Correa, I. R..

22 records · Page 2Linked to original sources

bioRxiv2019

Non-destructive enzymatic deamination enables single molecule long read sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single base resolution.

The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite treated DNA we applied a new enzymatic deamination approach, termed EM-seq (Enzymatic Methyl-seq) to long-range sequencing technologies. Our methodology, named LR-EM-seq (Long Range Enzymatic Methyl-seq) preserves the integrity of DNA allowing long-range methylation profiling of 5-mC and 5-hmC over several kilobases of genomic DNA. When applied to known differentially methylated regions (DMR), LR-EM-seq achieves phasing of over 5 kb resulting in broader and better defined DMRs compared to previously reported. This result demonstrated the importance of phasing methylation for biologically relevant questions and the applicability of LR-EM-seq for long range epigenetic analysis at single molecule and single nucleotide resolution.

genomics↗

bioRxiv2019

In vitro characterization of the yeast DcpS, a scavenger mRNA decapping enzyme

Eukaryotic mRNAs are modified at their 5 end early during transcription by the addition of N7-methylguanosine (m7G), which forms the \"cap\" on the first 5 nucleotide. Identification of the 5 nucleotide on mRNA is necessary for determination of the Transcription Start Site (TSS). We explored the effect of various reaction conditions on the activity of the yeast scavenger mRNA decapping enzyme DcpS (yDcpS) and examined decapping of 30 chemically distinct cap structures varying the state of methylation, sugar, phosphate linkage, and base composition on 25mer RNA oligonucleotides. Contrary to the generally accepted belief that DcpS enzymes only decap short oligonucleotides, we found that yDcpS efficiently decaps RNA transcripts as long as 1400 nucleotides. Further, we validated the application of yDcpS for enriching capped RNA using a strategy of specifically tagging the 5 end of capped RNA by first decapping and then recapping it with an affinity-tagged guanosine nucleotide.

molecular biology↗

bioRxiv2018

Agonist binding affinity determines palmitoylation of the glucagon-like peptide-1 receptor and its functional interaction with plasma membrane nanodomains in pancreatic beta cells

The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes and obesity, is known to undergo palmitoylation by covalent ligation of an acyl chain to cysteine 438 in its carboxyl-terminal tail. Work with other GPCRs indicates that palmitoylation can be dynamically regulated to allow receptors to partition into plasma membrane nanodomains that act as signaling hotspots. Here, we demonstrate that the palmitoylated state of the GLP-1R is increased by agonist binding, leading to its segregation and clustering into plasma membrane signaling nanodomains before undergoing internalization in a clathrin-dependent manner. Both GLP-1R signaling and trafficking are modulated by strategies targeting nanodomain segregation and cluster formation, including depletion of cholesterol or expression of a palmitoylation-defective GLP-1R mutant. Differences in receptor binding affinity exhibited by biased GLP-1R agonists, and modulation of binding kinetics with the positive allosteric modulator BETP, influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable.

cell biology↗

bioRxiv2017

Using high-throughput barcode sequencing to efficiently map connectomes

The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision--a \"connectome\"--is based on imaging methods that are slow, labor-intensive and expensive. Here we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence--an RNA \"barcode\"--which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and then joined into a form suitable for sequencing. Although at present the inefficiency in our hands of barcode joining precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost.

neuroscience↗