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Eukaryotic mRNA Processing
Unlike prokaryotes which have one RNA polymerase that makes all classes of RNA molecules, eukaryotic cells have three types of RNA polymerase (called RNA pol I, RNA pol II, and RNA pol III), and each type of RNA is made by its own polymerase:
RNA polymerase I makes ribosomal RNA (rRNA)
RNA polymerase II makes messenger RNA (mRNA)
RNA polymerase III makes transfer RNA (tRNA)
Moreover, RNAs are made in the nucleus of a eukaryotic cell, but function in protein synthesis in the cytoplasm. Unlike prokaryotic mRNAs, eukaryotic mRNAs undergo extensive modifications after synthesis by RNA polymerase II. These changes include capping,polyadenylation, and splicing.
Capping
Modification of the 5'-ends of eukaryotic mRNAs is called capping. The cap consists of a methylated GTP linked to the rest of the mRNA by a 5' to 5' triphosphate "bridge"(Figure 28.30). Capping occurs very early during the synthesis of eukaryotic mRNAs, even before mRNA molecules are finished being made by RNA polymerase II. Capped mRNAs are very efficiently translated by ribosomes to make proteins. In fact, some viruses, such as poliovirus, prevent capped cellular mRNAs from being translated into proteins. This enables poliovirus to take over the protein synthesizing machinery in the infected cell to make new viruses.
Polyadenylation
Modification of the 3'-ends of eukaryotic mRNAs is called polyadenylation (Figure BR). Polyadenylation is the addition of several hundred A nucleotides to the 3' ends of mRNAs. All eukaryotic mRNAs destined to get a poly A tail (note: most, but not all, eukaryotic mRNAs get such a tail) contain the sequence AAUAAA about 11-30 nucleotides upstream to where the tail is added. AAUAAA is recognized by an endonuclease that cuts the RNA, allowing the tail to be added by a specific enzyme:polyA polymerase.
Splicing
Eukaryotic genes are often interrupted by sequences that do not appear in the final RNA. The intervening sequences that are removed are called introns. The process by which introns are removed is referred to assplicing. The sequences remaining after the splicing are called exons. All of the different major types of RNA in a eukaryotic cell can have introns. Although most higher eukaryotic genes have introns, some do not. Higher eukaryotes tend to have a larger percentage of their genes containing introns than lower eukaryotes, and the introns tend to be larger as well. The pattern of intron size and usage roughly follows the evolutionary tree, but this is only a general tendency. The humantitin gene has the largest number of exons (178), the longest single exon (17,106 nucleotides) and the longest coding sequence (80,781 nucleotides = 26,927 amino acids). The longest primary transcript, however, is produced by the dystrophin gene (2.4 million nucleotides).
RNA-DNA Hybridization Reveals Spliced-out Introns
RNA splicing was discovered during analysis of adenovirus mRNA synthesis. In these studies, the abundant viral mRNA encoding the major virion capsid protein, called hexon, was isolated by gel electrophoresis of cytoplasmic polyadenylated RNA. To map the region of the viral DNA coding for hexon mRNA, researchers hybridized the isolated mRNA to the coding strand and the RNA-DNA hybrid was visualized in the electron microscope (Figure BL). Three loops of single-stranded DNA (A, B, and C) were observed; these correspond to the three introns in the hexon gene. Since these intron sequences in the viral genomic DNA are not present in mature hexon nRNA, they loop out between the exon sequences that hybridize to their complementary sequences in the mRNA.
Similar analyses of hybrids between RNA isolated from the nuclei of infected cells and viral DNA revealed RNAs that were coliner withe the viral DNA (primarly transcripts) and RNAs with one or two of the introns removed (processing intermediates). These results, together with the findings that the 5' cap and 3' poly-A tail of mRNA precoursors are retained in mature cytoplasmic mRNAs, led to the realization that introns are removed from primary transcripts as exons are spliced together. For short transcription units, RNA splicing usually follows cleavage and polyadenylation of the 3' end of the primary transcript. But for long transcription units containing multiple exons, splicing of exons in the nascent RNA sometimes begins before transcription of the gene is complete.
Splice Site in Pre-mRNAs Exhibit Short, Conserved Sequences
