Clinical prospects and research strategies of long non-coding RNA encoding micropeptides / 浙江大学学报·医学版

Long non-coding RNAs (lncRNAs) which are usually thought to have no protein coding ability, are widely involved in cell proliferation, signal transduction and other biological activities. However, recent studies have suggested that short open reading frames (sORFs) of some lncRNAs can encode small functional peptides (micropeptides). These micropeptides appear to play important roles in calcium homeostasis, embryonic development and tumorigenesis, suggesting their potential as therapeutic targets and diagnostic biomarkers. Currently, bioinformatic tools as well as experimental methods such as ribosome mapping and in vitro translation are applied to predict the coding potential of lncRNAs. Furthermore, mass spectrometry, specific antibodies and epitope tags are used for validating the expression of micropeptides. Here, we review the physiological and pathological functions of recently identified micropeptides as well as research strategies for predicting the coding potential of lncRNAs to facilitate the further research of lncRNA encoded micropeptides.

Increasing evidence for the presence of alternative proteins in human tissues and cell lines

Recent findings coming from human proteome research employing mass-spectrometry and ribosomal profiling methods have provided evidence for the translation of non-annotated coding sequence (CDSs) into alternative proteins (APs). The presence of APs in many human tissues and cell lines may become an important issue in genome sciences, especially in cancer genomics where the frequency of alternative proteins seems to be 10-fold higher than normal tissues. Finding new proteins can impact medical research by filling gaps in known molecular pathways or revealing new molecular markers and therapeutic targets. Among the cellular processes possibly involved in protein diversity, alternative splicing (AS) is the most cited, and it consists of an often-regulated mechanism that generates different mRNAs from the same gene, contributing to the functional diversity of mammalian cells. In the past, evidence for AS from multi-exon genes have come mainly from expression sequence tag (EST) data; only recently has mass-spectrometry (MS) been used to investigate the translation of alternative transcripts. Exploration of human MS data has detected tens to hundreds of alternative proteins in normal tissues, and thousands in cancer cell lines, suggesting that alternative proteins may have an important role in cancer. Analysis of MS data has revealed a vastly diverse AP repertoire, with some of this diversity being exclusively detected in cancer cells. Proteomic characterization of 20 breast cancer cell lines revealed a surprising 1,860 protein variants resulting from AS. Among these, 4 AP are clearly involved in cancer. A truncated variant of the NF- kB p65 subunit, a truncated form of the focal adhesion kinase PTK2 and two CD47 transmembrane receptor protein variants. Until now, little is known about the functional differences between these variants. Another cellular mechanism that possibly creates protein diversity is the alternative usage of translation initiation site (TIS). Detection of TIS is made possible by the Ribosome Profiling (RP) method. The principle of this technique is to capture mRNA translation by freezing the actively translating ribosomes onto transcripts, and then separating them by ultracentrifugation. Recently, RP was applied to mouse embryonic fibroblast cells and human HEK293 cells. The results revealed that the majority of mRNAs contain more than one translation initiation site (TIS), with more than 50% of the detected TISs mapping to alternative ORFs. In this review, we present a list of human alternative proteins validated by small and large-scale experimental methods. We also highlight that APs are probably not a secondary product of inaccurate splicing or translational process and most likely play an important role in the tumorigenic process. Thus, APs constitutes a promising research line for basic and clinical aspects of cancer (AU)

Ribosome Profiling for Biomarker Discovery / 日本補完代替医療学会誌

It has been well known that protein level is estimated by the expression level of its mRNA. However, it is also argued that correlation between mRNA abundance and protein levels is weaker than previously thought. Recently a newly developed technique called ribosome profiling has drawn attention as a drastic countermeasure to improve the weak correlation. Here it is discussed that weak association of protein and mRNA levels seen in genome-wide analysis of gene expression such as microarray is attributable to post-transcriptional regulation including translational inhibition. This review further discusses how these issues are resolved by ribosome profiling and also addresses a possibility of biomarker discovery derived from this technique.<br>