Part of RNA processing in eukaryotes is addition of the 5′ mRNA Cap, which is essential for mRNA stability, transport, and translation (by guiding ribosomal assembly at the RNA’s 5′ end). To ensure that only RNAP II transcripts are capped, the RNAP II CTD is phosphorylated, and the phosphorylated CTD recruits capping enzymes. After ~25-30 nucleotides have been transcribed and the nascent pre-mRNA is emerging from RNAP, the first transcribed nucleotide (the 5′ end) is bound via a 5′-5′ triphosphate linkage to a 7-methylguanosine. The 2’-hydroxyls of the first two transcribed nucleotides are also often methylated.

The Cap binds the nucleic Cap Binding Complex which guides splicing, RNA stabilization and transport into the cytoplasm. Once in the cytoplasm, the Cap binds the eIF4 complex, which helps initiate translation via binding the mRNA to the assembling ribosome, and defining the AUG start codon. By defining a single start codon, the Cap ensures that the mRNA encodes the same polypeptide whenever translated. This is a key difference from prokaryotic mRNAs, which are often polycystronic.

Cap Binding Complex

The Cap Binding Complex (CBC) is a nuclear protein with two subunits: the 20kD (CBP20) subunit binds Cap; 80kD (CBP80) subunit engages cofactors. The Cap Binding Complex is needed for RNA splicing, stabilizing the RNA, and transport out of the nucleus.

eIF4E

Once in the cytoplasm, the fully processed mRNA exchanges CBC for another Cap-binding protein: the E subunit of eukaryotic Initiation Factor 4 (eIF4E). The eIF4 complex delivers the mRNA to the small ribosomal subunit and helps identify the start codon during translation initiation.

Eukaryotes encode long precursor RNA transcripts, which are spliced, polyadenylated and capped to produce a messenger RNA that can be translated.

Just as transcription initiation and capping are coupled, transcription termination and the 3’ cleavage and polyadenylation reactions are functionally linked. The Cleavage/Polyadenylation reaction is carried out the polyadenylation complex, a large multi-protein complex assembled from CPSF, CStF, CF1, and CF2. Some pre-mRNA’s have multiple poly(a) signals, and cleavage and polyadenylation at these different signals can include or exclude various 3′ terminal exons.

Coupling 3′ Cleavage & Transcription Termination

What Is the Spliceosome?

The spliceosome is a hugely massive complex that catalyzes excision of introns and ligation of exons. In addition to many non-snRNP subunits, the spliceosome consists of 5 small nuclear ribonucleoproteins (snRNPs) that assemble stepwise onto each intron. The snRNPs are small and are composed of: a small nuclear RNA (snRNA) with a specific sequence and secondary structure; and some common and some specific proteins.

The five snRNPs of the spliceosome: U1, U2, U4 and U5 are transcribed by RNAP II and have a TriMethyl G Cap; U6 is transcribed by RNAP III and has an unusual Cap. In the U4/U6 Di-snRNP and the U4/U5/U6 Tri-snRNP, the U4 and U6 snRNAs are base-paired to each other. This interaction is later disrupted in the formation of the active spliceosome.

Remember that the splice sites do not always perfectly match the consensus sequences. Thus, the base-pairing interactions between the snRNAs and the pre-mRNA are not always the same. The interactions of U1 with the 5’ splice site and U2 with the branchpoint were proven by creating mutant splice sites that disrupted pairing with the snRNA and inhibited splicing. Compensating mutations in the snRNA that restored complementarity (base-pairing) with the splice site restored splicing.

Spliceosome Assembly

Most metazoan genes have multiple exons that must be carefully excised (from the pre-mRNA) and then ligated (to form the mRNA). This RNA splicing occurs in the nucleus, and upon its completion the mRNA is exported to the cytoplasm for translation. Exon definition complexes and spliceosomes begin to assemble during transcription. Some of these complexes interact with RNAP II’s CTD, and some introns are thus excised before transcription has even terminated. There is no apparent order in which introns get spliced). Introns do no excise in any particular order, and active transcription (or its termination) is not needed for splicing to occur. However, the rate of transcription elongation through an intron can strongly affect what splice sites are chosen and thus indirectly couple transcription and splicing. Exon definition complexes and spliceosomes begin assembling during RNA synthesis; interaction with RNAP II’s CTD leads to excision of some introns before transcription has even terminated).

Whether a gene has one or many introns, each is excised via the same two isonergetic transesterification reactions:

Intron Definition

Introns have sequences that directs the splicing apparatus during RNA splicing, part of RNA processing:

Some mitochondrial, protist and archaeal genes have autocatalytic self-splicing introns (as opposed to nuclear spliceosome-dependent introns). These introns fold into specific secondary and tertiary structures that catalyze their own excision, without proteins. There are two families of these introns — Group I and and Group II — and Group II autocatalytic introns are thought to operate with a similar mechanism to the spliceosome catalytic center.

Spinal Muscular Atrophy is the most common genetic cause of infant death, occurring in 1 in 6,000-10,000 births. This is an autosomal recessive disorder caused by mutations in the SMN1 gene, encoding a protein involved in snRNP maturation. Humans have undergone an SMN gene duplication generating the SMN2 gene. Even though it encodes an identical protein, the presence of SMN2 cannot compensate for the loss of SMN1 in spinal muscular atrophy, because its splicing is abnormal and it does not produce functional protein.

Splicing targeted therapies are being tested for Spinal Muscular Atrophy. A single C to T mutation in an exonic splicing enhancer of SMN2 exon 7 causes exon skipping and production of a truncated protein from 80% of the SMN2 mRNA. If drugs or other therapies could be found that increase exon 7 splicing of SMN2, one could potentially treat the disease even without correcting the SMN1 defect.

Nearly all disease genes have mutant alleles which affect splicing of the mRNA, rather than protein coding. Exon skipping can arise via mutations in the 5′ splice site, the 3′ splice site, and/or exonic or intronic splicing enhancers. Activation of cryptic splice sites (thus shortening the exon) can arise via mutated exonic splicing enhancers. Examples of diseases due to mRNA splicing errors include Familial Isolated Growth Hormone Deficiency (mutations in the GH-1 gene), Cystic Fibrosis (mutations in the CFTR gene), Spinal Muscular Atrophy and some thalassemias (mutations in the β-globin gene that activate a cryptic 3′ splice site).

Does gene number correlate with complexity? As shown to the rightt, it obviously does not. Rather, alternative splicing correlates with complexity. 95% of Human genes are known to exhibit alternative splicing. Complex Transcription Units use alternative splicing to produce more than one type of mRNA. Patterns of RNA spliciing can combine to produce a dizzying array of alternatively spliced isoforms: the Slo or Bk channel has over 500 isoforms; the neurexin protein has 2,346 isoforms; the para-sodium channel has 1,536 isoforms; the Drosophila DSCAM Receptor can potentially be made in 38,016 different spliced isoforms. Somatic sexual development in Drosophila is controlled by a cascade of splicing factors each regulating the splicing of genes downstream in the pathway. And cochlear hair cells are tuned to respond to different frequencies via alternative RNA splicing.

There are many examples of regulation in mammals where important changes in gene activity are regulated by alternative splicing. These systems use pre-mRNA binding proteins to enhance or repress particular splicing choices. These proteins have different types, numbers, and arrangements of RNA binding domains, and other domains that can be involved in protein/protein interactions. The RNA binding domains target the proteins to specific short sequence elements adjacent to sites of regulation. Alternative splicing is especially common in the mammalian nervous system, where it is used to diversify many proteins important for neuronal development and function. Small cassette exons create short peptide inserts that determine precise changes in ligand binding, electrophysiology, or subcellular targeting.