Key Concepts of Section 10.1

• In the nucleus of eukaryotic cells, pre-mRNAs are associated with hnRNP proteins and processed by 5′ capping, 3′ cleavage and polyadenylation, and splicing before being transported to the cytoplasm (see Figure 10-2).

• Shortly after transcription initiation, capping enzymes associate with the carboxy-terminal domain (CTD) of RNA polymerase II, phosphorylated multiple times at serine 5 of the heptapeptide repeat by TFIIH during transcription initiation. These enzymes then rapidly add the 5′ cap to the nascent transcript when it reaches a length of about 25 nucleotides. Other RNA-processing factors involved in RNA splicing and in 3′ cleavage and polyadenylation associate with the CTD when it is phosphorylated at serine 2 of the heptapeptide repeat, increasing the rate of transcription elongation. Consequently, transcription does not proceed at a high rate until RNA-processing factors become associated with the CTD, where they are poised to interact with the nascent pre-mRNA as it emerges from the surface of the polymerase.

• Five different snRNPs interact via base pairing with one another and with pre-mRNA to form the spliceosome (see Figure 10-11). This very large ribonucleoprotein complex catalyzes two transesterification reactions that join two exons and remove the intron as a lariat structure, which is subsequently degraded (see Figure 10-8).

• SR proteins that bind to exonic splicing enhancer sequences in exons are critical in defining exons in the large pre-mRNAs of higher organisms. A network of interactions between SR proteins, snRNPs, and splicing factors forms a cross-exon recognition complex that specifies correct splice sites (see Figure 10-13).

• The snRNAs in the spliceosome are thought to have an overall tertiary structure similar to that of group II self-splicing introns.

• For long transcription units in higher organisms, splicing of exons usually begins as the pre-mRNA is still being formed. Cleavage and polyadenylation to form the 3′ end of the mRNA occur after the poly(A) cleavage site is transcribed.

• In most protein-coding genes, a conserved AAUAAA polyadenylation signal lies slightly upstream from a poly(A) site where cleavage and polyadenylation occur. A GU- or U-rich sequence downstream from the poly(A) site contributes to the efficiency of cleavage and polyadenylation.

• A multiprotein complex that includes poly(A) polymerase (PAP) carries out the cleavage and polyadenylation of a pre-mRNA. A nuclear poly(A)-binding protein, PABPN1, stimulates addition of A residues by PAP and stops their addition once the poly(A) tail reaches about 250 residues (see Figure 10-15).

• Excised introns and RNA downstream from the cleavage/polyadenylation site are degraded primarily by exosomes, multiprotein complexes that contain an internal 3′→5′ exonuclease. Exosomes also degrade improperly processed pre-mRNAs.