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Related Concept Videos

Alternative RNA Splicing02:18

Alternative RNA Splicing

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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.
There are five types of alternative RNA splicing that vary in the ways the pre-mRNA segments are removed or retained in the mature mRNA. The first...
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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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Chromatin Structure Regulates pre-mRNA Processing02:41

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In eukaryotic cells, nascent mRNA transcripts need to undergo many post-transcriptional modifications to reach the cell cytoplasm and translate into functional proteins. For a long time, transcription and pre-mRNA processing were considered two independent events that occur sequentially in the cell. However, it has now been well established that transcription and pre-mRNA processing are two simultaneous processes that are precisely regulated inside the cell.
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Pre-mRNA Processing: Modification of pre-mRNA Ends01:35

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In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
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pre-mRNA Processing02:01

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In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
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Related Experiment Video

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Using the E1A Minigene Tool to Study mRNA Splicing Changes
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RNA Trans-Splicing Targeting Endogenous β-Globin Pre-Messenger RNA in Human Erythroid Cells.

Naoya Uchida1, Kareem N Washington2, Brian Mozer3

  • 11 Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH) , Bethesda, Maryland.

Human Gene Therapy Methods
|March 8, 2017
PubMed
Summary
This summary is machine-generated.

Researchers developed RNA trans-splicing to target sickle cell disease by replacing mutated beta-globin exon 1. This gene therapy approach successfully produced trans-spliced RNA in human erythroid cells, offering a potential treatment strategy.

Keywords:
RNA trans-splicinglentiviral vectorβ-globin gene

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Area of Science:

  • Molecular Biology
  • Gene Therapy
  • Hematology

Background:

  • Sickle cell disease is a genetic blood disorder caused by a point mutation in the beta-globin gene.
  • Current therapeutic strategies aim to correct the genetic defect or its consequences.
  • RNA trans-splicing presents a novel approach to gene correction by replacing mutated gene segments.

Purpose of the Study:

  • To develop and evaluate RNA trans-splicing targeting beta-globin pre-messenger RNA in human erythroid cells.
  • To identify optimal binding domains for efficient trans-splicing.
  • To establish a foundation for trans-splicing-based gene therapy for sickle cell disease.

Main Methods:

  • Screening of random beta-globin sequences to identify optimal binding domains targeting intron 2.
  • Construction of lentiviral vectors encoding RNA trans-splicing molecules with selected binding domains and gamma-globin cDNA.
  • Evaluation of trans-splicing in CD34+ cell-derived erythroid cells from healthy individuals.

Main Results:

  • Efficient lentiviral transduction was achieved (9.7-15.3 vector copy numbers).
  • The targeted trans-spliced RNA product, incorporating endogenous beta-globin exon 3 and gamma-globin, was detected.
  • Trans-splicing efficiency was enhanced to 0.07-0.09% using longer binding domains and the intron 2 5' splice site.

Conclusions:

  • Efficient binding domains for RNA trans-splicing were identified through comprehensive screening.
  • Detectable levels of trans-splicing for endogenous beta-globin RNA were achieved in human erythroid cells.
  • The developed methods support the advancement of trans-splicing directed gene therapy for sickle cell disease.