Quantifying full-length circular RNAs in cancer
Ken Hung-On YuChristina Huan ShiBo WangSavio Ho-Chit ChowGrace Tin‐Yun ChungRaymond Wai Ming LungKe‐En TanYat‐Yuen LimAnna Chi‐Man TsangKwok Wai LoKevin Y. Yip
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Abstract:
Circular RNAs (circRNAs) are abundantly expressed in cancer. Their resistance to exonucleases enables them to have potentially stable interactions with different types of biomolecules. Alternative splicing can create different circRNA isoforms that have different sequences and unequal interaction potentials. The study of circRNA function thus requires knowledge of complete circRNA sequences. Here we describe psirc, a method that can identify full-length circRNA isoforms and quantify their expression levels from RNA sequencing data. We confirm the effectiveness and computational efficiency of psirc using both simulated and actual experimental data. Applying psirc on transcriptome profiles from nasopharyngeal carcinoma and normal nasopharynx samples, we discover and validate circRNA isoforms differentially expressed between the two groups. Compared with the assumed circular isoforms derived from linear transcript annotations, some of the alternatively spliced circular isoforms have 100 times higher expression and contain substantially fewer microRNA response elements, showing the importance of quantifying full-length circRNA isoforms.Keywords:
Circular RNA
Splicing factor
Exonic splicing enhancer
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Abstract The beneficial influence of E2 in the maintenance of healthy bone is well recognized. However, the way in which the actions of this hormone are mediated is less clearly understood. Western blot analysis of ERα in osteoblasts clearly demonstrated that the well characterized 66-kDa ERα was only one of the ERα isoforms present. Here we describe a 46-kDa isoform of ERα, expressed at a level similar to the 66-kDa isoform, that is also present in human primary osteoblasts. This shorter isoform is generated by alternative splicing of an ERα gene product, which results in exon 1 being skipped with a start codon in exon 2 used to initiate translation of the protein. Consequently, the transactivation domain AF-1 of this ERα isoform is absent. Functional analysis revealed that human (h)ERα46 is able to heterodimerize with the full-length ERα and also with ERβ. Further, a DNA-binding complex that corresponds to hERα46 is detectable in human osteoblasts. We have shown that hERα46 is a strong inhibitor of hERα66 when they are coexpressed in the human osteosarcoma cell line SaOs. As a functional consequence, proliferation of the transfected cells is inhibited when increasing amounts of hERα46 are cotransfected with hERα66. In addition to human bone, the expression of the alternatively spliced ERα mRNA variant is also detectable in bone of ERα knockout mice. These data suggest that, in osteoblasts, E2 can act in part through an ERα isoform that is markedly different from the 66-kDa receptor. The expression of two ERα protein isoforms may account, in part, for the differential action that estrogens and estrogen analogs have in different tissues. In particular, the current models of the action of estrogens should be reevaluated to take account of the presence of at least two ERα protein isoforms in bone and perhaps in other tissues.
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Alternative splicing of pre-mRNA is a key mechanism for increasing the complexity of proteins in humans, causing a diversity of expression of transcriptomes and proteomes in a tissue-specific manner. Alternative splicing is regulated by a variety of splicing factors. However, the changes and errors of splicing regulation caused by splicing factors are strongly related to many diseases, something which represents one of this study’s main interests. Further understanding of alternative splicing regulation mediated by cellular factors is also a prospective choice to develop specific drugs for targeting the dynamic RNA splicing process. In this review, we firstly concluded the basic principle of alternative splicing. Afterwards, we showed how splicing isoforms affect physiological activities through specific disease examples. Finally, the available treatment methods relative to adjusting splicing activities have been summarized.
Proteome
Splicing factor
Minigene
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Protein isoform
Peptide mass fingerprinting
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Alternative splicing is a regulated process whereby one gene can generate multiple mRNA isoforms susceptible to be translated into protein isoforms of various functions. Several publications report the aberrant expression of splicing isoforms in cancer cells and tissues. However, in most cases, their function remains to be established. In this review article, I will discuss the molecular tool available to perform isoform-specific functional genomics, the methodologies to quantify their effectiveness and the resulting isoform-specific phenotype in human cancer cell lines.
Functional Genomics
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NF-YA, the regulatory subunit of the trimeric CCAAT-binding transcription factor NF-Y, is present in vertebrates in two major alternative spliced isoforms: NF-YAl and NF-YAs, differing for the presence of exon-3. NF-YAx, a third isoform without exon-3/-5, was reported only in human neuronal cells and tumors. These events affect the Trans-Activation Domain. We provide here evidence for the expression of NF-YAx and for the existence of a new isoform, NF-YAg, skipping only exon-5. These isoforms are abundant in Aves, but not in reptiles, and are the prevalent transcripts in the initial phases of embryo development in chicken. Finally, we analyzed NF-YAg and NF-YAx amino acid sequence using AlphaFold: absence of exon-5 denotes a global reduction of β-stranded elements, while removal of the disordered exon-3 sequence has limited effects on TAD architecture. These data identify an expanded program of NF-YA isoforms within the TAD in Aves, implying a role during early development.
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During pre-mRNA splicing events, introns are removed from the pre-mRNA, and the remaining exons are connected together to form a single continuous molecule. Alternative splicing is a common mechanism for the regulation of gene expression in eukaryotes. More than 90% of human genes are known to undergo alternative splicing. The most common type of alternative splicing is exon skipping, which is also known as cassette exon. Other known alternative splicing events include alternative 5' splice sites, alternative 3' splice sites, intron retention, and mutually exclusive exons. Alternative splicing events are controlled by regulatory proteins responsible for both positive and negative regulation. In this review, we focus on neuronal splicing regulators and discuss several notable regulators in depth. In addition, we have also included an example of splicing regulation mediated by the RBFox protein family. Lastly, as previous studies have shown that a number of splicing factors are associated with neuronal diseases such as Alzheime's disease (AD) and Autism spectrum disorder (ASD), here we consider their importance in neuronal diseases wherein the underlying mechanisms have yet to be elucidated.
Exon skipping
Exonic splicing enhancer
Minigene
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Splice site mutation
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