A Small RNA Isolation and Sequencing Protocol and Its Application to Assay CRISPR RNA Biogenesis in Bacteria.
2018; 8 (4)
Next generation high-throughput sequencing has enabled sensitive and unambiguous analysis of RNA populations in cells. Here, we describe a method for isolation and strand-specific sequencing of small RNA pools from bacteria that can be multiplexed to accommodate multiple biological samples in a single experiment. Small RNAs are isolated by polyacrylamide gel electrophoresis and treated with T4 polynucleotide kinase. This allows for 3' adapter ligation to CRISPR RNAs, which don't have pre-existing 3'-OH ends. Pre-adenylated adapters are then ligated using T4 RNA ligase 1 in the absence of ATP and with a high concentration of polyethylene glycol (PEG). The 3' capture step enables precise determination of the 3' ends of diverse RNA molecules. Additionally, a random hexamer in the ligated adapter helps control for potential downstream amplification bias. Following reverse-transcription, the cDNA product is circularized and libraries are prepared by PCR. We show that the amplified library need not be visible by gel electrophoresis for efficient sequencing of the desired product. Using this method, we routinely prepare RNA sequencing libraries from minute amounts of purified small RNA. This protocol is tailored to assay for CRISPR RNA biogenesis in bacteria through sequencing of mature CRISPR RNAs, but can be used to sequence diverse classes of small RNAs. We also provide a fully worked example of our data processing pipeline, with instructions for running the provided scripts.
View details for PubMedID 29600253
View details for PubMedCentralID PMC5870890
An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline.
2016; 166 (2): 343-357
Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here, we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-base pair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure.
View details for DOI 10.1016/j.cell.2016.05.072
View details for PubMedID 27374334
- An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline CELL 2016; 166 (2): 343-357
Translation readthrough mitigation
2016; 534 (7609): 719-?
A fraction of ribosomes engaged in translation will fail to terminate when reaching a stop codon, yielding nascent proteins inappropriately extended on their C termini. Although such extended proteins can interfere with normal cellular processes, known mechanisms of translational surveillance are insufficient to protect cells from potential dominant consequences. Here, through a combination of transgenics and CRISPR?Cas9 gene editing in Caenorhabditis elegans, we demonstrate a consistent ability of cells to block accumulation of C-terminal-extended proteins that result from failure to terminate at stop codons. Sequences encoded by the 3? untranslated region (UTR) were sufficient to lower protein levels. Measurements of mRNA levels and translation suggested a co- or post-translational mechanism of action for these sequences in C. elegans. Similar mechanisms evidently operate in human cells, in which we observed a comparable tendency for translated human 3? UTR sequences to reduce mature protein expression in tissue culture assays, including 3? UTR sequences from the hypomorphic ?Constant Spring? haemoglobin stop codon variant. We suggest that 3? UTRs may encode peptide sequences that destabilize the attached protein, providing mitigation of unwelcome and varied translation errors.
View details for DOI 10.1038/nature18308
View details for Web of Science ID 000378676000044
View details for PubMedID 27281202
View details for PubMedCentralID PMC5054982
Suppression of insulin production and secretion by a decretin hormone.
2015; 21 (2): 323-333
Decretins, hormones induced by fasting that suppress insulin production and secretion, have been postulated from classical human metabolic studies. From genetic screens, we identified Drosophila Limostatin (Lst), a peptide hormone that suppresses insulin secretion. Lst is induced by nutrient restriction in gut-associated endocrine cells. limostatin deficiency led to hyperinsulinemia, hypoglycemia, and excess adiposity. A conserved 15-residue polypeptide encoded by limostatin suppressed secretion by insulin-producing cells. Targeted knockdown of CG9918, a Drosophila ortholog of Neuromedin U receptors (NMURs), in insulin-producing cells phenocopied limostatin deficiency and attenuated insulin suppression by purified Lst, suggesting CG9918 encodes an Lst receptor. NMUR1 is expressed in islet ? cells, and purified NMU suppresses insulin secretion from human islets. A human mutant NMU variant that co-segregates with familial early-onset obesity and hyperinsulinemia fails to suppress insulin secretion. We propose Lst as an index member of an ancient hormone class called decretins, which suppress insulin output.
View details for DOI 10.1016/j.cmet.2015.01.006
View details for PubMedID 25651184
View details for PubMedCentralID PMC4349554
Direct assessment of hepatic mitochondrial oxidative and anaplerotic fluxes in humans using dynamic 13C magnetic resonance spectroscopy.
2014; 20 (1): 98?102
Despite the central role of the liver in the regulation of glucose and lipid metabolism, there are currently no methods to directly assess hepatic oxidative metabolism in humans in vivo. By using a new (13)C-labeling strategy in combination with (13)C magnetic resonance spectroscopy, we show that rates of mitochondrial oxidation and anaplerosis in human liver can be directly determined noninvasively. Using this approach, we found the mean rates of hepatic tricarboxylic acid (TCA) cycle flux (VTCA) and anaplerotic flux (VANA) to be 0.43 ± 0.04 ?mol g(-1) min(-1) and 0.60 ± 0.11 ?mol g(-1) min(-1), respectively, in twelve healthy, lean individuals. We also found the VANA/VTCA ratio to be 1.39 ± 0.22, which is severalfold lower than recently published estimates using an indirect approach. This method will be useful for understanding the pathogenesis of nonalcoholic fatty liver disease and type 2 diabetes, as well as for assessing the effectiveness of new therapies targeting these pathways in humans.
View details for DOI 10.1038/nm.3415
View details for PubMedID 24317120
View details for PubMedCentralID PMC3947269