While these regions may correspond to regulatory sequences, the biological significance of having many introns or having very long introns in a gene is unclear. It is possible that introns slow down gene expression because it takes longer to transcribe pre-mRNAs with lots of introns. Alternatively, introns may be nonfunctional sequence remnants left over from the fusion of ancient genes throughout evolution. This is supported by the fact that separate exons often encode separate protein subunits or domains.
For the most part, the sequences of introns can be mutated without ultimately affecting the protein product. All introns in a pre-mRNA must be completely and precisely removed before protein synthesis. If the process errs by even a single nucleotide, the reading frame of the rejoined exons would shift, and the resulting protein would be dysfunctional.
The presence of the cap protects the mRNA from degradation 3. During splicing, regions of mRNA sequences not expressed in proteins introns are excised and the remaining exons are joined together in a molecular machine termed the spliceosome4.
In the final step of RNA processing, called polyadenylation, several adenosine nucleotides are added to the 3' end of the mRNA, which helps to prevent degradation and promotes export from the nucleus to the cytoplasm 4.
After processing, export to the cytoplasm occurs through nuclear pore complexes. Molecular Cell Biology. New York: W. Chapter 11, RNA processing, nuclear transport, and post-transcriptional control. The spliceosome contains a specific set of U-rich small nuclear ribonucleoproteins or snRNPs. U1 5'- site recognition U2 branch site recognition U4 forms base paired complex and acts with U6 U5 3'- junction binding of U4-U6 complex U6 complex with U4 makes up the spliceosome transesterase.
The common spliceosome recognizes introns starting with 5'-GU and ending in AG-3'. More recently, a subclass of spliceosome has been found to recognize introns with 5'-AU and AC-3' ends. Tarn and Steitz, Incidence of introns increases with developmental complexity of the organism: Yeast has few introns, with well conserved and consistent splice and branch site sequences.
Higher organisms often have multiple introns within a single pre-mRNA, poorly defined splice and branch site sequences, and a more complex regulatory system controlling their selection. Splicing may be constitutive meaning that the same introns are always identified and spliced out from a pre-mRNA, resulting in translation to yield a single protein product.
However higher organisms make extensive use of alternative splicing to generate functionally different isoforms of a protein , which are expressed in particular states of differentiation or development. Regulatory mechanisms must determine which splice sites are selected. In addition to the snRNPs which consist of RNA and specific associated proteins a number of accessory protein factors are involved in various stages of the splicing reaction.
An additional protein factor, BBP branch binding protein binds in the region of the branchpoint A. This exposed ribose OH acts as the nucleophile attacking the 5'-splice site. When the sequence at the branchpoint deviates from the consensus, associated protein factors such as U2AF are needed to promote complex formation. Exons contain elements called exonic enhancers which are targets for binding SR and related RRM containing proteins.
An array of protein factors, e. SC35, bind in a cooperative manner between cap and first splice site to define its location. Other SR proteins bridge the intron gapfrom U1 70k to facilitate U2AF binding, and establish branch point and 3' splice site. Once the U2 complex is in place, SR proteins link up to the next 5' splice site, to continue the process. Thus the pattern of splice sites is established progressively from the 5' cap towards the 3' end , and the spliceosome does not select intron targets for splicing at random.
In metazoans, certain members of the hnRNP h eterogeneous n uclear ribonucleotprotein class bind to sites in particular in the introns. Splice site specificity is reasonably conserved across species, allowing expression of transgenes. Occasionally splice sites may be misread, for example when wild type Green Fluorescent Protein is expressed in higher plants, the polypeptide may be disrupted by misinterpretation of a coding sequence as a plant specific splicing site.
Alternative splicing Variation in splice site selection can result in expression of different, developmentally specific isoforms of a polypeptide from a single gene. In addition, initiation factors involved in protein synthesis recognize the cap to help initiate translation by ribosomes. The poly A tail protects the mRNA from degradation, aids in the export of the mature mRNA to the cytoplasm, and is involved in binding proteins involved in initiating translation.
This cleavage is done by an endonuclease-containing protein complex that binds to an AAUAAA sequence upstream of the cleavage site and to a GU-rich sequence downstream of the cut site. Eukaryotic genes are composed of exons, which correspond to protein-coding sequences ex -on signifies that they are ex pressed , and intervening sequences called introns int -ron denotes their int ervening role , which may be involved in gene regulation, but are removed from the pre-mRNA during processing.
Intron sequences in mRNA do not encode functional proteins. The discovery of introns came as a surprise to researchers in the s who expected that pre-mRNAs would specify protein sequences without further processing, as they had observed in prokaryotes. The genes of higher eukaryotes very often contain one or more introns.
While these regions may correspond to regulatory sequences, the biological significance of having many introns or having very long introns in a gene is unclear. It is possible that introns slow down gene expression because it takes longer to transcribe pre-mRNAs with lots of introns.
Alternatively, introns may be nonfunctional sequence remnants left over from the fusion of ancient genes throughout evolution. This is supported by the fact that separate exons often encode separate protein subunits or domains. For the most part, the sequences of introns can be mutated without ultimately affecting the protein product.
All introns in a pre-mRNA must be completely and precisely removed before protein synthesis. If the process errs by even a single nucleotide, the reading frame of the rejoined exons would shift, and the resulting protein would be dysfunctional. The process of removing introns and reconnecting exons is called splicing.
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