Mass Spectrometry-Based Proteomics to Unveil the Non-coding RNA World

The interaction between non-coding RNAs (ncRNAs) and proteins is crucial for the stability, localization and function of the different classes of ncRNAs. Although ncRNAs, when embedded in various ribonucleoprotein (RNP) complexes, control the fundamental processes of gene expression, their biological functions and mechanisms of action are still largely unexplored. Mass Spectrometry (MS)-based proteomics has emerged as powerful tool to study the ncRNA world: on the one hand, by identifying the proteins interacting with distinct ncRNAs; on the other hand, by measuring the impact of ncRNAs on global protein levels. Here, we will first provide a concise overview on the basic principles of MS-based proteomics for systematic protein identification and quantification; then, we will recapitulate the main approaches that have been implemented for the screening of ncRNA interactors and the dissection of ncRNA-protein complex composition. Finally, we will describe examples of various proteomics strategies developed to characterize the effect of ncRNAs on gene expression, with a focus on the systematic identification of microRNA (miRNA) targets

Complex translational regulation of BACE1 involves upstream AUGs and stimulatory elements within the 5′ untranslated region

BACE1 is the protease responsible for the production of amyloid-β peptides that accumulate in the brain of Alzheimer's disease (AD) patients. BACE1 expression is regulated at the transcriptional, as well as post-transcriptional level. Very high BACE1 mRNA levels have been observed in pancreas, but the protein and activity were found mainly in brain. An up-regulation of the protein has been described in some AD patients without a change in transcript levels. The features of BACE1 5′ untranslated region (5′ UTR), such as the length, GC content, evolutionary conservation and presence of upstream AUGs (uAUGs), indicate an important regulatory role of this 5′ UTR in translational control. We demonstrate that, in brain and pancreas, almost all of the native BACE1 mRNA contains the full-length 5′ UTR. RNA transfection and in vitro translation show that translation is mainly inhibited by the presence of the uAUGs. We provide a mutational analysis that highlight the second uAUG as the main inhibitory element while mutations of all four uAUGs fully de-repress translation. Furthermore, we have evidence that a sequence within the region 222-323 of the BACE1 5′ UTR has a stimulatory effect on translation that might depend on the presence of trans-acting factors

MS-analysis of SILAC-labeled MYC-driven B lymphoma cells overexpressing miR-17-19b

Micro RNAs (miRNAs) are small non-coding RNAs, which dampen gene expression by repressing translation and/or inducing degradation of target-mRNAs. Although the role of miR-17-19b (a truncated version of miR-17-92 cluster) is well documented in MYC-driven B cell lymphomagenesis, little is known about the function of the cluster in the maintenance of full-blown lymphomas. We employed SILAC-based quantitative proteomics to identify miR-17-19b targets upon a mild overexpression of the cluster in B cell lymphomas, established from λ-MYC transgenic mice. The proteomics data described in detail in this study, whose follow up analysis with MaxQuant algorithm is part of the recent publication (Mihailovich et al., 2015) [1], are deposited to the ProteomeXchange Consortium via the PRIDE partner repository, with the accession code PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002810. Keywords: MiR-17-92, SILAC, Quantitative proteomics, Mass spectrometry, MiRNA targets, MYC, B cell lymphom

Complex translational regulation of BACE1 involves

upstream AUGs and stimulatory elements within the 5 0 untranslated regio

BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR

<p><b>Copyright information:</b></p><p>Taken from "Complex translational regulation of BACE1 involves upstream AUGs and stimulatory elements within the 5′ untranslated region"</p><p></p><p>Nucleic Acids Research 2007;35(9):2975-2985.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888809.</p><p>© 2007 The Author(s)</p> () Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. () Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. () RNase protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is γ-ATP labeled 50 bp DNA marker. () qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6)

Different regions of BACE1 5′ UTR affect translation in a positive or negative manner

<p><b>Copyright information:</b></p><p>Taken from "Complex translational regulation of BACE1 involves upstream AUGs and stimulatory elements within the 5′ untranslated region"</p><p></p><p>Nucleic Acids Research 2007;35(9):2975-2985.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888809.</p><p>© 2007 The Author(s)</p> () Schematic representation of mRNAs bearing different BACE1 5′ UTR deletion mutants and firefly luciferase as reporter gene. Black boxes represent uORFs, and arrows uAUGs. () RNA transfections of different deletion mutants in HeLa cell line together with control reporter mRNA. The results are presented in fold variation. Error bars denote the standard deviation from the mean of at least three independent experiments. The star indicates

UAUGs are responsible for low translational efficiency driven by BACE1 5′ UTR

<p><b>Copyright information:</b></p><p>Taken from "Complex translational regulation of BACE1 involves upstream AUGs and stimulatory elements within the 5′ untranslated region"</p><p></p><p>Nucleic Acids Research 2007;35(9):2975-2985.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888809.</p><p>© 2007 The Author(s)</p> () Schematic representation of reporter mRNAs bearing mutations of uAUGs. Arrows represent uAUGs or UUGs when mutated; black boxes symbolize uORFs. () Transfection of reporter mRNAs into HeLa cell line. mRNA was used to normalize for transfection efficiency. Results are presented in fold variation, and the error bars present the mean of at least three independent experiments. Star indicates