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RNA Splicing01:32

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Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
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Alternative RNA splicing is the regulated splicing of exons and introns to produce different mature mRNAs from a single pre-mRNA. Unlike in constitutive splicing where a single gene produces a single type of mRNA, alternative splicing allows an organism to produce multiple proteins from a single gene and plays an important role in protein diversity.
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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R...
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The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
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Use of Alu Element Containing Minigenes to Analyze Circular RNAs
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Use of Alu Element Containing Minigenes to Analyze Circular RNAs

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Efficient backsplicing produces translatable circular mRNAs.

Yang Wang1, Zefeng Wang2

  • 1Institute of Cancer Stem Cell, the Second Affiliated Hospital, Cancer Center, Dalian Medical University, Dalian 116044, China Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

RNA (New York, N.Y.)
|December 3, 2014
PubMed
Summary
This summary is machine-generated.

Circular RNAs (circRNAs) are abundant but poorly understood. This study shows backsplicing efficiently generates circRNAs in diverse eukaryotes, which can be translated into functional proteins.

Keywords:
alternative splicingbacksplicingcircular RNAsplicing factorstranslation

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A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
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Area of Science:

  • Molecular Biology
  • Genetics
  • RNA Biology

Background:

  • Circular RNAs (circRNAs) are prevalent in the human transcriptome, yet their functions and biogenesis mechanisms remain largely elusive.
  • Sequence analysis indicates circRNAs arise from exons spliced in reverse order (backsplicing), but the underlying molecular mechanisms are not well understood.

Purpose of the Study:

  • To investigate the mechanisms of backsplicing and circRNA generation in eukaryotic cells.
  • To determine if circRNAs can be translated into functional proteins and how their translation is regulated.

Main Methods:

  • Construction of a single-exon minigene with split Green Fluorescent Protein (GFP) to study backsplicing.
  • Experimental validation in human and Drosophila cells to assess circRNA production and translation.
  • Analysis of the role of intronic complementarity, splicing factors, and cis-elements in regulating backsplicing.

Main Results:

  • Efficient backsplicing and circRNA production were observed in both human and Drosophila cells using the minigene system.
  • Complementary introns forming double-stranded RNA structures enhance backsplicing, but are not strictly required.
  • Backsplicing is regulated by general splicing factors and cis-elements through distinct mechanisms compared to canonical splicing.
  • The generated circRNAs are translatable into functional proteins.
  • Poly-adenosine or poly-thymidine sequences in the 3' untranslated region (UTR) inhibit circRNA translation, unlike in linear mRNA.

Conclusions:

  • Backsplicing is an efficient and conserved mechanism for generating circular mRNAs across diverse eukaryotes.
  • circRNAs can be translated into functional proteins, with translation regulation differing from linear mRNAs.
  • Understanding circRNA biogenesis and function opens new avenues in RNA biology and potential therapeutic applications.