snoRNA-facilitated protein secretion revealed by transcriptome-wide snoRNA target identification.

Small nucleolar RNAs (snoRNAs) are non-coding RNAs known for guiding RNA modifications, including 2'-O-methylation (N) and pseudouridine (Ψ). While snoRNAs may also interact with other RNAs, such as mRNA, the full repertoire of RNAs targeted by snoRNA remains elusive due to the lack of effective technologies that identify snoRNA targets transcriptome wide. Here, we develop a chemical crosslinking-based approach that comprehensively detects cellular RNA targets of snoRNAs, yielding thousands of previously unrecognized snoRNA-mRNA interactions in human cells and mouse brain tissues.

PTPN2 copper-sensing relays copper level fluctuations into EGFR/CREB activation and associated CTR1 transcriptional repression.

Fluxes in human copper levels recently garnered attention for roles in cellular signaling, including affecting levels of the signaling molecule cyclic adenosine monophosphate. We herein apply an unbiased temporal evaluation of the signaling and whole genome transcriptional activities modulated by copper level fluctuations to identify potential copper sensor proteins responsible for driving these activities. We find that fluctuations in physiologically relevant copper levels modulate EGFR signal transduction and activation of the transcription factor CREB.

Small-molecule inhibition of the METTL3/METTL14 complex suppresses neuroblastoma tumor growth and promotes differentiation.

The N-methyladenosine (mA) RNA modification is an important regulator of gene expression. mA is deposited by a methyltransferase complex that includes methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). High levels of METTL3/METTL14 drive the growth of many types of adult cancer, and METTL3/METTL14 inhibitors are emerging as new anticancer agents.

KARR-seq reveals cellular higher-order RNA structures and RNA-RNA interactions.

RNA fate and function are affected by their structures and interactomes. However, how RNA and RNA-binding proteins (RBPs) assemble into higher-order structures and how RNA molecules may interact with each other to facilitate functions remain largely unknown. Here we present KARR-seq, which uses N-kethoxal labeling and multifunctional chemical crosslinkers to covalently trap and determine RNA-RNA interactions and higher-order RNA structures inside cells, independent of local protein binding to RNA.

Sequencing of N-methyl-deoxyadenosine at single-base resolution across the mammalian genome.

Although DNA N-methyl-deoxyadenosine (6mA) is abundant in bacteria and protists, its presence and function in mammalian genomes have been less clear. We present Direct-Read 6mA sequencing (DR-6mA-seq), an antibody-independent method, to measure 6mA at base resolution. DR-6mA-seq employs a unique mutation-based strategy to reveal 6mA sites as misincorporation signatures without any chemical or enzymatic modulation of 6mA.

Profiling of RNA-binding protein binding sites by in situ reverse transcription-based sequencing.

RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA targets are still lacking, especially when the interaction is dynamic or samples are limited. Here we present an assay of reverse transcription-based RBP binding site sequencing (ARTR-seq), which relies on in situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites.

PTPN2 copper-sensing rapidly relays copper level fluctuations into EGFR/CREB activation and associated transcriptional repression.

Fluxes in human intra- and extracellular copper levels recently garnered attention for roles in cellular signaling, including affecting levels of the signaling molecule cyclic adenosine monophosphate (cAMP). We herein applied an unbiased temporal evaluation of the whole-genome transcriptional activities modulated by fluctuations in copper levels to identify the copper sensor proteins responsible for driving these activities. We found that fluctuations in physiologically-relevant copper levels rapidly modulate EGFR/MAPK/ERK signal transduction and activation of the transcription factor cAMP response element-binding protein (CREB).

mA-SAC-seq for quantitative whole transcriptome mA profiling.

N-methyladenosine (mA) is the most abundant mRNA modification in mammalian cells, regulating many physiological processes. Here we describe a method for base-resolution, quantitative mA sequencing in the whole transcriptome. The enzyme and small-molecule cofactor used in this protocol are prepared by recombinant protein expression and organic synthesis, respectively.

Keth-seq for transcriptome-wide RNA structure mapping.

RNA secondary structure is critical to RNA regulation and function. We report a new N-kethoxal reagent that allows fast and reversible labeling of single-stranded guanine bases in live cells. This N-kethoxal-based chemistry allows efficient RNA labeling under mild conditions and transcriptome-wide RNA secondary structure mapping.

Divergent Entry to Gelsedine-Type Alkaloids: Total Syntheses of (-)-Gelsedilam, (-)-Gelsenicine, (-)-Gelsedine, and (-)-Gelsemoxonine.

The gelsedine-type alkaloids possess a common oxabicyclo[3.2.2]nonane core and spiro- N-methoxyindolinone moiety along with a diversely functionalized heterocycle embedded in the compact framework.

N-heterocyclic carbene-catalyzed stereoselective cascade reaction: synthesis of functionalized tetrahydroquinolines.

The first N-heterocyclic carbene-catalyzed stereoselective aza-Michael-Michael-lactonization cascade reaction of 2'-aminophenylenones and 2-bromoenals for the construction of chiral functionalized tetrahydroquinolines with three consecutive stereogenic centers has been achieved in high yields (up to 98%) with excellent diastereo- (>25:1) and enantioselectivities (up to 98.7% ee).