Cross-platform proteomics signatures of extreme old age.

In previous work we used a Somalogic platform targeting approximately 5000 proteins to generate a serum protein signature of centenarians that we validated in independent studies that used the same technology. We set here to validate and possibly expand the results by profiling the serum proteome of a subset of individuals included in the original study using liquid chromatography tandem mass spectrometry (LC-MS/MS). Following pre-processing, the LC-MS/MS data provided quantification of 398 proteins, with only 266 proteins shared by both platforms. At 1% FDR statistical significance threshold, the analysis of LC-MS/MS data detected 44 proteins associated with extreme old age, including 23 of the original analysis. To identify proteins for which associations between expression and extreme-old age were conserved across platforms, we performed inter-study conservation testing of the 266 proteins quantified by both platforms using a method that accounts for the correlation between the results. From these tests, a total of 80 proteins reached 5% FDR statistical significance, and 26 of these proteins had concordant pattern of gene expression in whole blood. This signature of 80 proteins points to blood coagulation, IGF signaling, extracellular matrix (ECM) organization, and complement cascade as important pathways whose protein level changes provide evidence for age-related adjustments that distinguish centenarians from younger individuals.

Isha is a su(Hw) mRNA-binding protein required for gypsy insulator function.

Chromatin insulators are DNA-protein complexes localized throughout the genome capable of establishing independent transcriptional domains. It was previously reported that the Drosophila su(Hw) mRNA physically associates with the gypsy chromatin insulator protein complex within the nucleus and may serve a noncoding function to affect insulator activity. However, how this mRNA is recruited to the gypsy complex is not known. Here, we utilized RNA-affinity pulldown coupled with mass spectrometry to identify a novel RNA-binding protein, Isha (CG4266), that associates with su(Hw) mRNA in vitro and in vivo. Isha harbors a conserved RNA recognition motif and RNA Polymerase II C-terminal domain-interacting domain (CID). We found that Isha physically interacts with total and elongating Polymerase II and associates with chromatin at the 5' end of genes in an RNA-dependent manner. Furthermore, ChIP-seq analysis reveals Isha overlaps particularly with the core gypsy insulator component CP190 on chromatin. Depletion of Isha reduces enhancer-blocking and barrier activities of the gypsy insulator and disrupts the nuclear localization of insulator bodies. Our results reveal a novel factor Isha that promotes gypsy insulator activity that may act as a nuclear RNA-binding protein adapter for su(Hw) noncoding mRNA.

Activation of PKR by short stem-loop RNAs containing single-stranded arms.

Protein kinase R (PKR) is a central component of the innate immunity antiviral pathway and is activated by dsRNA. PKR contains a C-terminal kinase domain and two tandem dsRNA binding domains. In the canonical activation model, binding of multiple PKR monomers to dsRNA enhances dimerization of the kinase domain, leading to enzymatic activation. A minimal dsRNA of 30 bp is required for activation. However, short (∼15 bp) stem-loop RNAs containing flanking single-stranded tails (ss-dsRNAs) are capable of activating PKR. Activation was reported to require a 5'-triphosphate. Here, we characterize the structural features of ss-dsRNAs that contribute to activation. We have designed a model ss-dsRNA containing 15-nt single-stranded tails and a 15-bp stem and made systematic truncations of the tail and stem regions. Autophosphorylation assays and analytical ultracentrifugation experiments were used to correlate activation and binding affinity. PKR activation requires both 5'- and 3'-single-stranded tails but the triphosphate is dispensable. Activation potency and binding affinity decrease as the ssRNA tails are truncated and activation is abolished in cases where the binding affinity is strongly reduced. These results indicate that the single-stranded regions bind to PKR and support a model where ss-dsRNA induced dimerization is required but not sufficient to activate the kinase. The length of the duplex regions in several natural RNA activators of PKR is below the minimum of 30 bp required for activation and similar interactions with single-stranded regions may contribute to PKR activation in these cases.