Figure 6–25 The pre-mRNA splicing reaction. (A) In the first step, a specific adenine nucleotide in the intron sequence (indicated in red ) attacks the 5ʹ splice site and cuts the sugar-phosphate backbone of the RNA at this point. The cut 5ʹ end of the intron becomes covalently linked to the adenine nucleotide, as shown in detail in (B), thereby creating a loop in the RNA molecule. The released free 3ʹ-OH end of the exon sequence then reacts with the start of the next exon sequence, joining the two exons together and releasing the intron sequence in the shape of a lariat.
The two exon sequences thereby become joined into a continuous coding sequence. The released intron sequence is eventually broken down into single nucleotides, which 5˜ 3˜ are recycled.
TRANSCRIPTION, SPLICING, AND 3˜ CLEAVAGE/POLYADENYLATION
The mechanism of pre-mRNA splicing shown in Figure 6–24 requires that the splicing machinery recognize three portions of the precursor RNA molecule: the 5ʹ splice site, the 3ʹ splice site, and the branch point in the intron sequence that forms the base of the excised lariat. Not surprisingly, each site has a consensus nucleotide sequence that is similar from intron to intron and provides the cell with cues for where splicing is to take place (Figure 6–27). However, these consensus sequences are relatively short and can accommodate extensive sequence variability; as we shall see shortly, the cell incorporates additional types of information to ultimately choose exactly where, on each RNA molecule, splicing is to take place.
The high variability of the splicing consensus sequences presents a special challenge for scientists attempting to decipher genome sequences. Introns range in size from about 10 nucleotides to over 100,000 nucleotides, and choosing the precise borders of each intron is a difficult task even with the aid of powerful computers. The possibility of alternative splicing compounds the problem of predicting protein sequences solely from a genome sequence. This difficulty is one of the main barriers to identifying all of the genes in a complete genome sequence, and it is one of the primary reasons why we know only the approximate number of different proteins produced by the human genome.
RNA Splicing Is Performed by the Spliceosome
Unlike the other steps of mRNA production we have discussed, key steps in RNA splicing are performed by RNA molecules rather than proteins. Specialized RNA molecules recognize the nucleotide sequences that specify where splicing is to occur and also catalyze the chemistry of splicing. These RNA molecules are relatively short (less than 200 nucleotides each), and there are five of them, U1, U2, U4, U5, and U6. Known as snRNAs (small nuclear RNAs), each is complexed with at least seven protein subunits to form an snRNP (small nuclear ribonucleoprotein).
–°–°–°AG GURAGU –°– –°– °YURAC – ....°– YYYYYYYYNCAG G –°–°–° 5˜3˜ portion of a primary transcript exon 1 intron exon 2 portion of mRNA exon 1 exon 2
Figure 6–26 Alternative splicing of the α-tropomyosin gene from rat.
α-Tropomyosin is a coiled-coil protein (see Figure 3–9) that carries out several tasks, most notably the regulation of contraction in muscle cells. The primary transcript can be spliced in different ways, as indicated in the figure, to produce distinct mRNAs, which then give rise to variant proteins. Some of the splicing patterns are specific for certain types of cells. For example, the α-tropomyosin made in striated muscle is different from that made from the same gene in smooth muscle. The arrowheads in the top part of the figure mark the sites where cleavage and poly-A addition form the 3ʹ ends of the mature mRNAs.