High-throughput and site-specific identification of 2'-O-methylation sites using ribose oxidation sequencing (RibOxi-seq).

Ribose methylation (2'-O-methylation, 2'-OMe) occurs at high frequencies in rRNAs and other small RNAs and is carried out using a shared mechanism across eukaryotes and archaea. As RNA modifications are important for ribosome maturation, and alterations in these modifications are associated with cellular defects and diseases, it is important to characterize the landscape of 2'-O-methylation. Here we report the development of a highly sensitive and accurate method for ribose methylation detection using next-generation sequencing. A key feature of this method is the generation of RNA fragments with random 3'-ends, followed by periodate oxidation of all molecules terminating in 2',3'-OH groups. This allows only RNAs harboring 2'-OMe groups at their 3'-ends to be sequenced. Although currently requiring microgram amounts of starting material, this method is robust for the analysis of rRNAs even at low sequencing depth.

An Ultraconserved Element (UCE) controls homeostatic splicing of ARGLU1 mRNA.

Arginine and Glutamate-Rich protein 1 (ARGLU1) is a protein whose function is poorly understood, but may act in both transcription and pre-mRNA splicing. We demonstrate that the ARGLU1 gene expresses at least three distinct RNA splice isoforms - a fully spliced isoform coding for the protein, an isoform containing a retained intron that is detained in the nucleus, and an isoform containing an alternative exon that targets the transcript for nonsense mediated decay. Furthermore, ARGLU1 contains a long, highly evolutionarily conserved sequence known as an Ultraconserved Element (UCE) that is within the retained intron and overlaps the alternative exon. Manipulation of the UCE, in a reporter minigene or via random mutations in the genomic context using CRISPR/Cas9, changed the splicing pattern. Further, overexpression of the ARGLU1 protein shifted the splicing of endogenous ARGLU1 mRNA, resulting in an increase in the retained intron isoform and nonsense mediated decay susceptible isoform and a decrease in the fully spliced isoform. Taken together with data showing that functional protein knockout shifts splicing toward the fully spliced isoform, our data are consistent with a model in which unproductive splicing complexes assembled at the alternative exon lead to inefficient splicing and intron retention.

Long noncoding RNAs in imprinting and X chromosome inactivation.

The field of long noncoding RNA (lncRNA) research has been rapidly advancing in recent years. Technological advancements and deep-sequencing of the transcriptome have facilitated the identification of numerous new lncRNAs, many with unusual properties, however, the function of most of these molecules is still largely unknown. Some evidence suggests that several of these lncRNAs may regulate their own transcription in cis, and that of nearby genes, by recruiting remodeling factors to local chromatin. Notably, lncRNAs are known to exist at many imprinted gene clusters. Genomic imprinting is a complex and highly regulated process resulting in the monoallelic silencing of certain genes, based on the parent-of-origin of the allele. It is thought that lncRNAs may regulate many imprinted loci, however, the mechanism by which they exert such influence is poorly understood. This review will discuss what is known about the lncRNAs of major imprinted loci, and the roles they play in the regulation of imprinting.

Functionally defective germline variants of sialic acid acetylesterase in autoimmunity.

Sialic acid acetylesterase (SIAE) is an enzyme that negatively regulates B lymphocyte antigen receptor signalling and is required for the maintenance of immunological tolerance in mice. Heterozygous loss-of-function germline rare variants and a homozygous defective polymorphic variant of SIAE were identified in 24/923 subjects of European origin with relatively common autoimmune disorders and in 2/648 controls of European origin. All heterozygous loss-of-function SIAE mutations tested were capable of functioning in a dominant negative manner. A homozygous secretion-defective polymorphic variant of SIAE was catalytically active, lacked the ability to function in a dominant negative manner, and was seen in eight autoimmune subjects but in no control subjects. The odds ratio for inheriting defective SIAE alleles was 8.6 in all autoimmune subjects, 8.3 in subjects with rheumatoid arthritis, and 7.9 in subjects with type I diabetes. Functionally defective SIAE rare and polymorphic variants represent a strong genetic link to susceptibility in relatively common human autoimmune disorders.

Esterases and autoimmunity: the sialic acid acetylesterase pathway and the regulation of peripheral B cell tolerance.

The best studied mechanisms of B cell tolerance are receptor editing, clonal deletion and anergy. All of these mechanisms of B cell tolerance depend on the induction of signaling downstream of the B cell receptor by self-antigens. Another important and distinct mechanism of B cell tolerance involves the repression of antigen receptor signaling rather than its induction, utilizes the Lyn Src-family kinase, the SHP-1 tyrosine phosphatase, inhibitory members of the Siglec family, and a carbohydrate-modifying enzyme that is capable of negatively regulating B cell receptor activation known as sialic acid acetylesterase.