Addition of a 5′-cap: This is the first of the processing reactions for premRNA (Fig. 31.17). The cap is a 7-methylguanosine attached to the 5′terminal end of the mRNA through an unusual 5′→5′-triphosphate linkage that is resistant to most nucleases. Creation of the cap requires removal of the γ phosphoryl group from the 5′-triphosphate of the premRNA, followed by addition of guanosine monophosphate (from guanosine triphosphate) by the nuclear enzyme guanylyltransferase. Methylation of this terminal guanine occurs in the cytosol and is catalyzed by guanine-7-methyltransferase. S-Adenosylmethionine is the source of the methyl group (see p. 263). Additional methylation steps may occur. The addition of this 7-methylguanosine cap helps stabilize the mRNA and permits efficient initiation of translation (see p. 455).
2.
Addition of a 3′-poly-A tail: Most eukaryotic mRNA (with several exceptions, including those for the histones) have a chain of 40–250 adenylates (adenosine monophosphates) attached to the 3′-end (see Fig. 31.17). This poly-A tail is not transcribed from the DNA but rather is added by the nuclear enzyme, polyadenylate polymerase, using ATP as the substrate. The pre-mRNA is cleaved downstream of a consensus sequence, called the polyadenylation signal sequence (AAUAAA), found near the 3′-end of the RNA, and the poly-A tail is added to the new 3′end. Tailing terminates eukaryotic transcription. Tails help stabilize the mRNA, facilitate its exit from the nucleus, and aid in translation. After the mRNA enters the cytosol, the poly-A tail is gradually shortened.
3.
Splicing: Maturation of eukaryotic mRNA usually involves removal from the primary transcript of RNA sequences (introns or intervening sequences) that do not code for protein. The remaining coding (expressed) sequences, the exons, are joined together to form the mature mRNA. The process of removing introns and joining exons is called splicing. The molecular complex that accomplishes these tasks is known as the spliceosome. A few eukaryotic primary transcripts contain no introns (for example, those from histone genes). Others contain a few introns, whereas some, such as the primary transcripts for the α chains of collagen, contain >50 introns that must be removed.
a. Role of small nuclear RNA: In association with multiple proteins, uracil-rich snRNA form five small nuclear ribonucleoprotein particles (snRNP, or “snurp”) designated as U1, U2, U4, U5, and U6 that mediate splicing. They facilitate the removal of introns by forming base pairs with the consensus sequences at each end of the intron (Fig. 31.18). [Note: In systemic lupus erythematosus, an autoimmune disease, patients produce antibodies against their own nuclear proteins such as snRNP.] b.