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After the RNA molecule is produced by transcription (Part 9), the structure of the RNA is often modified prior to being translated into a protein product. These RNA modifications apply mainly to eukaryotic RNA transcripts.
The modifications to eukaryotic RNA transcripts include the following:
The remainder of Part 10 will focus on the first three RNA modifications: 5' end capping, 3' end polyadenylation, and RNA splicing.
The 5’ end of the pre-mRNA molecule is modified by the addition of a 7-methylguanosine (7-mG) nucleotide. The process of adding the 7-mG to the pre-mRNA is 5’ end capping. 5’ end capping is the first RNA modification, occurring as soon as the 5' end of the pre-mRNA emerges from RNA polymerase II during transcription. 5’ end capping (see Figure 10.2) involves the following enzymes:
The 7-mG cap on eukaryotic mRNAs has at least three functions. The 7-mG cap:
The 3’ end of the pre-mRNA is modified by the addition of a polyA tail, a string of approximately 250 adenine (A) nucleotides. The process of adding a polyA tail to the mRNA transcript (see Figure 10.3), called 3’ end polyadenylation, involves:
The polyA tail on the mRNA has at least three functions. The polyA tail functions to:
The 3’ end polyadenylation process occurs after 5’ end capping, but prior to RNA splicing. In fact, 3’ end polyadenylation assists in terminating transcription in eukaryotes by the torpedo model (see Part 9).
There are three general mechanisms used by eukaryotes to remove introns from RNA molecules. The group I and group II mechanisms are limited to certain types of eukaryotes or certain organelles within a eukaryotic cell. For example, the group I mechanism removes the introns found in ribosomal RNA (rRNA) molecules in certain protozoa. The group II mechanism removes the introns found in the mRNA and transfer RNA (tRNA) molecules produced by mitochondrial and chloroplast genes. The spliceosome mechanism is the major mechanism that is used to remove introns from pre-mRNA transcripts in the nucleus of eukaryotic cells.
Transcription of most structural genes in the nucleus of eukaryotic cells produces pre-mRNA molecules; the removal of the introns within these pre-mRNA molecules involves a large multi-subunit spliceosome complex. To remove introns from the pre-mRNA, the spliceosome binds to recognition sequences within the intron (see Figure 10.6). These intron recognition sequences include:
The spliceosome complex contains multiple subunits; these subunits are called small nuclear ribonucleoproteins or snRNPs (“snurps”). Each snRNP within the spliceosome complex is composed of a small nuclear RNA (snRNA) molecule that acts as an RNA enzyme (ribozyme) to remove the introns from the pre-mRNA molecule. snRNPs are also composed of proteins that function to stabilize snRNP structure.
The spliceosome splicing mechanism occurs as follows:
Introns were initially identified within eukaryotic genes by performing R-loop (hybridization) experiments. These R-loop experiments relied on separating the two DNA strands within a gene, allowing a mRNA molecule to form hydrogen bonds (hybridize) with the template DNA strand, and adding the coding strand DNA, which attempts to form hydrogen bonds with the template DNA strand. Finally, the resulting nucleic acid structure was examined in an electron microscope. Below are the results expected from two R-loop experiments, one experiment involving the pre-mRNA (before RNA modifications), the other experiment involving the mature mRNA (after RNA modifications).
Introns within genes can also be identified by comparing the length of a genomic DNA (gDNA) version of a gene to the complementary DNA (cDNA) version of the same gene. gDNA is the version of a gene found in the genome; the gDNA version of a gene contains both introns and exons. cDNA is produced in the laboratory by reverse transcription (see Part 8). Reverse transcription converts mature mRNA into a cDNA molecule using the viral enzyme reverse transcriptase. Since the cDNA molecule is produced from the mature mRNA, cDNA molecules contain exons but lack introns. The gDNA version of the gene, which contains introns, will be longer than the cDNA version of the same gene, which lacks introns.
The polymerase chain reaction (PCR) technique (see Part 8) can be used to make billions of copies of the gDNA and the cDNA versions of any gene of interest. The gDNA and cDNA PCR products are then separated by size using agarose gel electrophoresis (see Part 8). The size difference between the gDNA and the cDNA copy of the gene can be easily observed on an agarose gel (see Figure 3.2).
Alternative splicing involves splicing a single type of pre-mRNA molecule in different ways to produce multiple mature mRNA molecules (see Figure 10.9). Each of these mature mRNAs can then produce slightly different proteins upon translation. These distinct, yet related protein isoforms, all derived from a single gene, can have specialized functions. Alternative splicing is beneficial in that it allows eukaryotes to carry fewer genes in the genome, permitting a relatively small number of genes the flexibility to encode a vast array of proteins. In humans, it is estimated that 30–60% of the genes in the genome are alternatively spliced. As a result, the human genome, which contains approximately 23,000 structural genes, can produce at least ten times that number of unique protein products.
One example of alternative splicing involves the human α-tropomyosin gene, a gene involved in muscle contraction. The α-tropomyosin gene contains 14 exons and 13 introns. The α-tropomyosin gene contains two types of exons:
Alternative splicing is regulated by splicing factor proteins. These splicing factor proteins help the spliceosome choose which intron splice sites to cut during RNA splicing. Each cell type has a different collection of splicing factor proteins, allowing different RNA splicing patterns to occur in each cell type.
Here are some common alternative splicing patterns observed in eukaryotic cells:
Scientists are still learning the true complexity of alternative splicing. It appears that alternative splicing patterns are cell-type and developmental stage specific. Moreover, mutations often lead to aberrant splicing patterns. This aberrant splicing produces abnormal protein isoforms and in some cases, disease phenotypes.
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