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Intron retention and its impact on gene expression and protein diversity: A review and a practical guide

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Abstract Intron retention (IR) occurs when a complete and unspliced intron remains in mature mRNA. An increasing body of literature has demonstrated a major role for IR in numerous biological functions, including several that impact human health and disease. Although experimental technologies used to study other forms of mRNA splicing can also be used to investigate IR, a specialized downstream computational analysis is optimal for IR discovery and analysis. Here we provide a review of IR and its biological implications, as well as a practical guide for how to detect and analyze it. Several methods, including long read third generation direct RNA sequencing, are described. We have developed an R package, FakIR, to facilitate the execution of the bioinformatic tasks recommended in this review and a tutorial on how to fit them to users aims. Additionally, we provide guidelines and experimental protocols to validate IR discovery and to evaluate the potential impact of IR on gene expression and protein output. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Splicing Regulation/Alternative Splicing RNA Methods > RNA Analyses in vitro and In Silico
The many fates of mRNA with retained introns. In the nucleus, the primary transcript may either be completely spliced (1) and rapidly be exported to the cytoplasm (2) or it may be incompletely spliced to retain one or more introns (3). In response to a signal, it may then undergo further “delayed” splicing (4) and export (2) or it may be degraded in the nucleus (5). It may also be exported directly without further splicing (6). In the cytoplasm, all mRNAs undergo a pioneer round of translation (7). The fully spliced mRNA may be translated into a polypeptide (8). If the mRNA retains an intron that is in frame, it may be translated into a polypeptide that contains a novel internal domain encoded by the intron (9). If the mRNA retains an intron that contains an in‐frame stop codon, it is often degraded by nonsense mediated mRNA decay (NMD) (10). If NMD is avoided it may be translated into a truncated polypeptide with a C‐terminal domain encoded by the intron (11). The Cap binding proteins CBP20 and CBP80 that are present on the mRNA during the pioneer round of translation are replaced by EIF‐4E as translation proceeds in the cytoplasm. PABP is the poly‐A binding protein. Different isoforms of PABP bind poly‐A in the nucleus and cytoplasm. Many other proteins that are not shown are also bound to the mRNA and facilitate translation (Harvey et al., 2018)
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Screenshot of the GWIPS‐viz browser. Data displaying elongating Ribo‐Seq (red) and RNA‐Seq (green) data aggregated from 37 experiments. It shows the GWIPS‐viz generated Ribo‐seq (top panel) and RNA‐seq (bottom panel) data tracks of Nxf1 gene. One can clearly see the retention of intron 10 from Ribo‐seq track and that the Ribo‐seq peaks are present in the corresponding region. On the basis of this clear RPF coverage across the intron (top panel), it can be inferred that the Nxf1 transcript containing intron 10 is likely translated, as is known from the experimental evidence discussed in this review
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A generalized scheme of ribosome and polyribosome profiling protocols. Ribosome and polysome profiling protocols can be utilized to determine if IR‐mRNA discovered in cytoplasmic lysates are potentially translated. The panel on the left demonstrates experimental steps to produce ribosomal profiling or Ribo‐seq data. The panel on the right demonstrates key experimental steps of polysome profiling
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Overview of the procedure to detect IR events and differential IR from multiple sources and types of sequencing data. The flow diagram starts with either short‐read (second generation) mRNA‐seq data or long‐read (third generation) mRNA‐seq data which fundamentally determines which bioinformatic pipelines should be used. The fundamental steps of each pipeline are similar‐quality control of raw sequencing data followed by intron detection, filtration, and ultimately quantification/differential expression analysis
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RNA Methods > RNA Analyses In Vitro and In Silico
RNA Processing > Splicing Regulation/Alternative Splicing
RNA Evolution and Genomics > Computational Analyses of RNA

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