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RNA Splicing

RNA Splicing

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...

RNA Splicing

RNA Splicing

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...

Pre-mRNA Processing: Modification of pre-mRNA Ends

Pre-mRNA Processing: Modification of pre-mRNA Ends

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.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a cap to the 5' end of the growing transcript. In this process, a 5' phosphate is replaced by modified guanosine that has a methyl group attached (7-methyl guanosine). This 5' cap helps the cell...

pre-mRNA Processing

pre-mRNA Processing

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.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...

Pre-mRNA Processing

Pre-mRNA Processing

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.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...

Alternative RNA Splicing

Alternative RNA Splicing

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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A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

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Processing of multiple-intron-containing pretRNA.

Giuseppe D Tocchini-Valentini¹, Paolo Fruscoloni, Glauco P Tocchini-Valentini

¹Istituto di Biologia Cellulare, Consiglio Nazionale delle Ricerche, Campus A, Buzzati-Traverso, Via Ramarini 32, Monterotondo Scalo, 00016 Rome, Italy. gtocchini@ibc.cnr.it

Proceedings of the National Academy of Sciences of the United States of America

|November 14, 2009

Summary

This summary is machine-generated.

Multiple introns in archaeal tRNA genes are processed sequentially. This study demonstrates in vitro the sequential splicing of these complex pretRNAs by specific tRNA splicing endonucleases.

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Area of Science:

Molecular Biology
Biochemistry
Archaea Genomics

Background:

Computational studies suggest multiple, nested introns in certain archaeal tRNA genes.
These pretRNAs are predicted to fold into specific conformations (bulge-helix-bulge motifs) for sequential splicing.
tRNA splicing endonucleases are responsible for intron removal.

Purpose of the Study:

To investigate the in vitro processing of archaeal pretRNAs containing multiple introns.
To determine if tRNA splicing endonucleases can process these complex substrates.
To compare the activity of homotetrameric (Methanocaldococcus jannaschii) and heterotetrameric (Sulfolobus solfataricus) enzymes.

Main Methods:

In vitro splicing assays using purified tRNA splicing endonucleases.
Enzymes used: Methanocaldococcus jannaschii (METJA) homotetrameric enzyme and Sulfolobus solfataricus (SULSO) heterotetrameric enzyme.
Analysis of pretRNA processing based on predicted substrate recognition motifs (bulge-helix-bulge and bulge-helix-loop).

Main Results:

Demonstrated in vitro processing of multiple-intron-containing pretRNA.
The SULSO enzyme showed broader substrate specificity, potentially cleaving variant structures.
The METJA enzyme's activity was consistent with recognition of the bulge-helix-bulge motif.

Conclusions:

The sequential splicing of multiple-intron-containing archaeal pretRNAs is feasible in vitro.
Different tRNA splicing endonuclease structures exhibit distinct substrate recognition capabilities.
This study provides experimental evidence for a predicted complex biological mechanism in archaea.