10.1 Processing of Eukaryotic Pre-mRNA
In this section, we take a closer look at how eukaryotic cells convert the initial primary transcript synthesized by RNA polymerase II into a functional mRNA. Three major events occur during the process: 5′ capping, 3′ cleavage and polyadenylation, and RNA splicing (Figure 10-2). Adding these specific modifications to the 5′ and 3′ ends of the pre-mRNA protects it from enzymes that quickly digest uncapped RNAs generated by RNA processing, such as spliced-out introns and RNA transcribed downstream from a polyadenylation site. Thus the 5′ cap and 3′ poly(A) tail distinguish pre-mRNA molecules from the many other kinds of RNAs in the nucleus (Table 10-1). Pre-mRNA molecules are bound by nuclear proteins that function in mRNA export to the cytoplasm. Prior to nuclear export, introns must be removed to generate the correct coding region of the mRNA. In higher eukaryotes, including humans, alternative splicing is intricately regulated in order to substitute different functional domains into proteins, producing a considerable expansion of the proteome of these organisms.
FIGURE 10-2 Overview of mRNA processing in eukaryotes. Shortly after RNA polymerase II initiates transcription at the first nucleotide of the first exon of a gene, the 5′ end of the nascent RNA is capped with 7-methylguanylate (step 1 ). Transcription by RNA polymerase II terminates at any one of multiple termination sites downstream from the poly(A) site, which is located at the 3′ end of the final exon. After the primary transcript is cleaved at the poly(A) site (step 2 ), a string of adenosine (A) residues is added (step 3 ). The poly(A) tail contains ~250 A residues in mammals, ~150 in insects, and ~100 in yeasts. For short primary transcripts with few introns, splicing (step 4 ) usually follows cleavage and polyadenylation, as shown. For large genes with multiple introns, introns are often spliced out of the nascent RNA during its transcription, before transcription of the gene is complete. Note that the 5′ cap and the sequence adjacent to the poly(A) tail are retained in mature mRNAs. The diagram shown represents processing of human β-globin RNA.
The pre-mRNA processing events of capping, polyadenylation, and splicing occur in the nucleus as the nascent mRNA precursor is being transcribed. Thus pre-mRNA processing is co-transcriptional. As the RNA emerges from the surface of RNA polymerase II, its 5′ end is immediately modified by the addition of the 5′ cap structure found on all mRNAs (see Figure 5-14). As the nascent pre-mRNA continues to emerge from the surface of the polymerase, it is immediately bound by members of a complex group of RNA-binding proteins that assist in RNA splicing and export of the fully processed mRNA through nuclear pore complexes into the cytoplasm. Some of these proteins remain associated with the mRNA in the cytoplasm, but most either remain in the nucleus or shuttle back into the nucleus shortly after the mRNA is exported to the cytoplasm. Cytoplasmic RNA-binding proteins are exchanged for the nuclear ones. Consequently, mRNAs never occur as free RNA molecules in the cell, but are always associated with proteins as ribonucleoprotein (RNP) complexes, first as nascent pre-mRNPs that are capped and spliced as they are transcribed. Then, following cleavage and polyadenylation, they are referred to as nuclear mRNPs. Following the exchange of proteins that accompanies export to the cytoplasm, they are called cytoplasmic mRNPs. Although we frequently refer to pre-mRNAs and mRNAs, it is important to remember that they are always associated with proteins as RNP complexes.