Splicing Activity Reconstitution
Splicing Activity Reconstitution - Related Videos
09:25
In vitro Reconstitution of the Active T. castaneum Telomerase
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Cited by 5 •
2011
Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
02:18
Alternative RNA Splicing
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2020
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...
01:32
RNA Splicing
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2019
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...
09:59
Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein
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Cited by 7 •
2015
Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.
07:05
Reconstitution of Membrane Proteins
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2023
Reconstitution is the process of returning an isolated biomolecule to its original form or function. This is particularly useful for studying membrane proteins, which enable important cellular functions and affect the behavior of nearby lipids. To study the function of purified membrane proteins in situ, they must be reconstituted by integrating them into an artificial lipid membrane. This video introduces membrane protein reconstitution concepts and related procedures, such as protein...
FAQs
These three pre-mRNA landmarks provide the positional information needed for accurate processing. Spliceosomal proteins and small nuclear ribonucleoproteins recognize the sites in the substrate, establishing the arrangement required for catalysis. Examining how changes in individual factors affect recognition or assembly helps distinguish defects in site selection from defects in later catalytic steps.
The reactions separate splicing into two chemically defined stages. First, cleavage and intron rearrangement produce an intron lariat, a looped intron intermediate. Second, the exons are ligated into a continuous RNA product. Reconstituted assays allow researchers to determine whether a component supports early assembly, catalytic progression, or completion of exon joining.
A defined system links an observed splicing outcome to the components deliberately included in the reaction. Researchers can therefore test how particular spliceosomal proteins or small nuclear ribonucleoproteins affect assembly, site recognition, or catalysis without relying only on the complexity of a cellular extract. This precision supports mechanistic analysis of factor function and regulation.
The assay follows a functional sequence in which spliceosomal components assemble on a pre-mRNA substrate before the RNA-processing reactions occur. ATP is present during this process, and the assembled machinery must recognize the relevant splice sites before producing the lariat and ligated exons. Comparing these stages helps identify where a factor acts in the pathway.
A typical reaction combines a pre-mRNA substrate with spliceosomal proteins and small nuclear ribonucleoproteins in the presence of ATP. These defined ingredients provide both the RNA target and the molecular machinery needed for assembly and catalysis. Adjusting which components are included enables direct testing of their contributions to splicing activity and regulation.
The approach is useful when researchers need to connect a mutation or compound exposure with a specific change in RNA processing. By examining spliceosome assembly, factor function, site recognition, or the formation of lariat and ligated-exon products, investigators can analyze how the perturbation affects the pathway. The same framework supports comparisons across regulatory conditions.
Results can clarify how individual factors contribute to spliceosome assembly, catalytic progression, and regulatory control. They can also support comparisons of RNA-processing pathways across evolutionary contexts. Because the reactions are reconstructed from defined components, the assay provides a biochemical framework for relating molecular mechanism to altered splicing associated with mutations or pharmacological interventions.