A Comparison of Line Blots, Enzyme-linked Immunosorbent, and RNA-immunoprecipitation Assays of Antisynthetase Antibodies in Serum Samples from 44 Patients.

Objective To determine the differences between anti-aminoacyl tRNA synthetase (ARS) antibodies among line blots, enzyme-linked immunosorbent assay (ELISA) anti-ARS tests, and RNA-immunoprecipitation (IP) assays. Methods Sera from patients with confirmed or suspected antisynthetase syndrome (ASS) that were positive for either the anti-ARS test or the line-blot assay were used to perform an RNA-IP assay and ELISA to detect individual anti-ARS antibodies. Results Among the 44 patients, 10 were positive only in line-blot assays, 6 were positive only in the anti-ARS test, and 28 were positive in both assays. We compared the accuracy of these assays against the gold standard RNA-IP assay. The κ coefficient was 0.23 in the line-blot assay, but this increased to 0.75 when the cut-off was increased from 1+ to 2+. The κ coefficient was 0.73 in the anti-ARS test. The κ coefficient was 0.85 for positivity in both assays. Patients with ASS that was positive in an RNA-IP assay more frequently had mechanic's hand (62.1% vs. 20%: p=0.031), myositis (51.7 vs. 10%: p=0.028) and more ASS symptoms than those who were positive only in line-blot assays (3.48 vs. 2.2: p=0.019). Conclusions Clinicians need to understand the features of each assay and determine diagnoses by also considering clinical presentations. Diagnoses should not be judged based only on the results of line-blot assays due to the risk of a misdiagnosis from false positives.

The Intriguing Conundrum of a Nonconserved Multifunctional Protein of Citrus Tristeza Virus That Interacts with a Viral Long Non-Coding RNA.

Citrus tristeza virus (CTV), the largest non-segmented plant RNA virus, has several peculiar features, among which is the production of a 5'-terminal long non-coding RNA (lncRNA) termed low-molecular-weight tristeza 1 (LMT1). In this study, we found that p33, a unique viral protein that performs multiple functions in the virus infection cycle, specifically binds LMT1, both in vivo and in vitro. These results were obtained through the expression of p33 under the context of the wild type virus infection or along with a mutant CTV variant that does not produce LMT1 as well as via ectopic co-expression of p33 with LMT1 in Nicotiana benthamiana leaves followed by RNA immunoprecipitation and rapid amplification of cDNA ends assays. Further experiments in which a recombinant p33 protein and an in vitro transcribed full-length LMT1 RNA or its truncated fragments were subjected to an electrophoretic mobility shift assay demonstrated that p33 binds to at least two distinct regions within LMT1. To the best of our knowledge, this is the first report of a plant virus protein binding to a lncRNA produced by the same virus. The biological significance of the interaction between these two viral factors is discussed.

High throughput platform to explore RNA-protein interactomes.

RNA binding proteins (RBPs) and RNA interaction is an emerging topic in molecular biology. Many reports showed that such interactions contribute to many cellular processes as well as disease development. Several standard in vitro and in vivo methods were developed to fulfill the needs of this RBP-RNA interaction study to explore their biological functions. However, these methods have their limitations in terms of throughput. In this review, we emphasize two important high throughput methods to studying RBP-RNA interactions, affinity purification and protein microarray. These methods have recently become robust techniques regarding their efficiency in systematically analyzing RBP-RNA interactions. Here, we provide technique overviews, strategies and applications of these methods during biological research. Although these technologies are just beginning to be explored, they will be most important methods in this study.

Screening for possible miRNA-mRNA associations in a colon cancer cell line.

MicroRNAs (miRNAs) are small non-coding RNAs mediating the regulation of gene expression in various biological contexts, including carcinogenesis. Here, we screened putative associations between 34, 45, and 103 miRNAs and 164, 391, and 81 mRNAs via Argonaute1 (Ago1) or Ago2 immunoprecipitation (IP) experiments in a colon cancer cell line. We used a combination of RIP Seq analysis. RNAs that were co-immunoprecipitated with Ago1 or Ago2 were used for massively parallel small RNA and mRNA sequencing. The detected miRNAs and mRNAs were further associated with one another based on in silico target predictions. Analysis of the putative associations indicated that, although Ago1 and Ago2 shared a similar repertory of miRNAs, the mRNAs possibly regulated by those miRNAs seemed different. The mRNAs detected with Ago1 IP were indicated to be frequently associated with genes having constitutive cellular functions, regulated by a smaller number of miRNAs, and appeared to receive more stringent translational regulation. In contrast, putative miRNA-mRNA associations detected with Ago2 IP appeared to be related to signal transduction genes, which had a larger number of possible miRNA binding sites. We then conducted a similar analysis using the colon cancer cells cultured under hypoxia and identified potential hypoxia-induced miRNA-mRNA associations, which included several well-characterized cancer-related genes as novel putative miRNA targets.