RNA Capping:

In eukaryotes, transcription is only the first step leading up to a mature mRNA which can be transported out of the nucleus to the cytoplasm of the cell for translation. Intron (noncoding) sequences must also be removed from the primary transcript and covalent modifications must be made at the ends of the mRNA. However, these events are coupled and can occur simultaneously during transcription. For example, an RNA cap is added and splicing of introns typically begins before transcription has been completed. This coupling is achieved through the RNA polymerase tail where proteins on the tail jump onto the nascent RNA molecule to begin processing it as soon as it emerges from the polymerase.

Eurkayrotic mRNA is modified in the nucleus with the addition of a methylated GTP to the 5′ end of the traanscript, called the 5′ cap, and a long chain of adenine residues to the 3′ end of the transcript, called the 3′ poly-A tail. When the transcript reaches about 20 nucleotides, it is modified by the addition of GTP to the 5′ PO4- group. The G in the GTP is also modifed by the addition of a methyl group, so it is often called a methyl-G cap. The transcript is cleaved downstream at a specific site (AAUAAA) while the polyemrease elongating and a series of 100-200 adenosine resiudes, the 3′ poly-A tail, is added to the mRNA. The enzyme responsible for this is poly-A polymerase.

RNA capping at the 5′ end is the first modification of eukaryotic pre-mRNAs. This cap is an unusual 5′ to 5′ linkage of 7-methylguanosine to the mRNA. The 5′ methyl cap helps to distinguish mRNAs from other RNA molecules present in the cell. For example, RNA polymerases I and III produce uncapped RNAs during transcription. The cap is also used to help the RNA to be properly processed and exported and will have an important role in the translation of mRNAs in the cytosol.

In humans, only 1-1.5% of the genome is devoted to the exons that encode proteins; 24% is devoted to the noncoding introns within which these exons are embedded. The primary transcript is but and put back together to produce the mature mRNA, in a process known as pre-mRNA splicing. It occurs in the nucleus prior to the export of the mRNA to the cytoplasm.

Splicing:

The introns-exon junctions are recognized by small nuclear ribonucleoprotein particles (snRNPs) or “snurps”): The snRNPs are complexes composed of snRNA and protein. These snRNPs cluster together with other associated proteins to form a larger complex called the spliceosome, which is respnsible for the splicing or remal of the introns. Introns all being with the same 2 base sequence and end with another 2 base sequence that tags them for removal. No rules govern the number of introns per gene or the sizes of introns and exons. Some genes have no introns; others have as many as 50. The sizes of exons range from a few nucleotides to 7500 nt and the sizes of introns are equally variable. Also, if eveyr gnee was spliced to include all exons, then the number of genes, transcripts and proteins would be euqal. But this is not the case. A single primary transcript can be spliced into different mRNA by using different sets of exons in a process called alternative splicing. It is estimated that up to 95 of human genes prodce multiple splce products.

RNA splicing involves the joining of two exons while removing an intron as a “lariat.” RNA splicing is performed mostly by snRNAs rather than proteins. These snRNPs form the core of a spliceosome which is a large assembly of RNA and protein molecules that performs pre-mRNA splicing. The process starts with a specific adenine nucleotide at a “branch point” in the intron sequence which attacks a 5′ splice site, cutting the sugar phosphate backbone of the RNA. The 5′ end of the intron becomes covalently linked to the A residue. The released 3′ OH end of the exon then reacts with the start of the next exon sequence, joining the 2 exons together and releasing the intron sequence in the shape of a lariat.

Generating the 3′ end of a eukaryotic mRNA is much more complicated than in bacteria where RNA polymerase simply stops at a termination signal and releases both the 3′ end of its transcript and the DNA template. In eukaryotes, there is a consensus sequence in the DNA which directs cleavage of the transcribed mRNA as well as polyadenylation. 2 proteins called CstF (cleavage stimulation Factor F) and CPSF (cleavage and polyadenylation specificity factor) are crucial for this process. First the RNA is cleaved and then another enzyme called poly-A polymerase adds about 200 A nucleotides to the 3′ end produced by the cleavage. Unlike the usual RNA polymerase, poly-A polymerase does not require a template.

Protein binding to a pre-mRNA is necessary not only for transcription of the mRNA, but is also necessary for transportation of the transcript out of the nucleus to the cytoplasm where translation will occur. Only if the proper set of proteins is bound to a mRNA will it be guided through the nuclear pore complex into the cytosol. Nuclear pore complexes are aqueous channel in the nuclear membrane that allow small molecules to diffuse. But macromolecules like mRNA need special processes to move them. Signals on the macromolecule determine whether it is exported and many of the proteins necessary for splicing are of key importance.

Regulated alternative splicing of pre-mRNA is a critical mechanisms by which functionally different proteins are generated from the same gene. Splice site selection and specificy are influenced by 5′ and 3′ splice sites located at the exon-intron boundaries of pre-mRNAs and by exonic splicing enhancer (ESE) and suppressor (ESS) elements. In general, the binding of serine/arginine rich proteins (SR proteins) to the ESEs enhances splicing and the binding to the ESSs by members of the heterogenoeous nuclear rebonucleoportein (hnRNP) family results in a supression of splicing. SR proteins stimulate the selection of intron-proximal 5′ splice sites in pre-mRNAs that contain 2 or more alternative 5′ splice sites, while hnRNPs have the opposite effect, promoting the selection of intron-distal 5′ spice sites.