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mRNA Processing in Eukaryotes
This Sqadia video is the demonstration of Post Transcriptional Gene Control. Control may be exerted as a primary transcript is processed in the nucleus, during export of an mRNA to the cytoplasm, or in the cytoplasm. Any one gene would likely be regulated by only one or a few of the possible control mechanisms. 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. For large genes with multiple introns, introns often are spliced out of the nascent RNA during its transcription. 5’ cap and sequence adjacent to the poly(A) tail are retained in mature mRNAs. Cleavage and polyadenylation specificity factor (CPSF) binds to the upstream AAUAAA poly(A) signal. CStF interacts with a downstream GU- or U-rich sequence and with bound CPSF, forming a loop in the RNA; binding of CFI and CFII help stabilize the complex. After 200–250 A residues have been added, PABPII signals PAP to stop polymerization. Two transesterification reactions result in splicing of exons in pre-mRNA, in the first reaction, the ester bond between the 5’ phosphorus of the intron and the 3’ oxygen of exon 1 is exchanged for an ester bond with the 2’ of the branch-site A residue. In the second reaction, the ester bond between the 5’ phosphorus of exon 2 and the 3’ oxygen of the intron is exchanged for an ester bond with the 3’ oxygen of exon 1, releasing the intron as a lariat structure and joining the two exons.
Splicing of Pre-mRNA
U2 snRNA base pairs with a sequence that includes the branch-point A, although this residue is not base-paired. Sequences that bind snRNP proteins are recognized by anti-Sm antibodies. Antisera from patients with the autoimmune disease systemic lupus erythematosus contain these antibodies. Such antisera have been useful in characterizing components of the splicing reaction. A mutation (A) in a pre-mRNA splice site that interferes with base-pairing to the 5’ end of U1 snRNA blocks splicing. After U1 and U2 snRNPs associate with the pre-mRNA, via base-pairing interactions a trimeric snRNP complex of U4, U5, and U6 joins the initial complex to form the spliceosome. The catalytic core catalyzes the first transesterification reaction. Following further rearrangements between the snRNPs, the second transesterification reaction joins the two exons. The excised lariat intron is converted into a linear RNA by a debranching enzyme. The correct 5’ GU and 3’ AG splice sites are recognized by splicing factors on the basis of their proximity to exons. When bound to ESEs, the SR proteins interact with one another and promote the cooperative binding. The RNA-protein cross-exon recognition complex activates the correct splice sites for RNA splicing. When the secondary structures of group II self-splicing introns and U snRNAs present in the spliceosome are compared, the similarity in their structures suggests that the spliceosomal snRNAs evolved from group II introns, with the trans-acting snRNAs being functionally analogous to the corresponding domains in group II introns.
Regulation of Pre-mRNA Processing
Because of alternative splicing of primary transcripts and cleavage at different poly(A) sites, different mRNAs may be expressed from the same gene in different cell types or at different developmental stages. Only female embryos produce functional Sxl protein. The cooperative binding of Tra protein and two SR proteins, Rbp1 and Tra2, activates splicing and cleavage/polyadenylation in dsx pre-mRNA in female embryos. Because Rbp1 and Tra2 cannot bind to the pre-mRNA in the absence of Tra, exon 4 is skipped in male embryos. The Slo protein contains seven transmembrane helices (S0–S6), which associate to form the K+ channel. Isoforms of the Slo channel, encoded by alternatively spliced mRNAs produced from the same primary transcript, open at different Ca2+ concentrations and thus respond to different frequencies. The apoB mRNA produced in the liver has the same sequence as the exons in the primary transcript. This mRNA is translated into apoB-100, which has two functional domains. In the apo-B mRNA produced in the intestine, the CAA codon in exon 26 is edited to a UAA stop codon.
Nuclear Transport of Cargo Proteins
In the cytoplasm, a free importin binds to the NLS of a cargo protein, forming a bimolecular cargo complex. In the case of a basic NLS, the adapter protein importin bridges the NLS and importin, forming a trimolecular cargo complex. A GTPase accelerating protein (GAP) associated with the cytoplasmic filaments of the NPC stimulates Ran to hydrolyze the bound GTP. This generates a conformational change causing dissociation from the importin. In the nucleoplasm, the protein exportin 1 binds cooperatively to the NES of the cargo protein to be transported and to Ran·GTP. Ran·GDP is transported through interaction with NTF2. Ran-GEF in the nucleoplasm then stimulates conversion of Ran·GDP to Ran·GTP. The large subunit of the mRNA-exporter contains three key domains: a middle domain (M) and a carboxyl domain (C), and an N-terminal region. The small subunit binds to the middle domain of the large subunit and contributes to the binding of FG repeats. Nascent RNA transcripts produced from the template DNA rapidly associate with proteins, forming hnRNPs. The gradual increase in size of the hnRNPs reflects the increasing length of RNA transcripts. Following processing of the pre-mRNA, the resulting ribonucleoprotein particle is referred to as an mRNP. As the mRNA enters the cytoplasm, it rapidly associates with ribosomes, indicating that the 5’ end passes through the NPC first.
Control of Transport and Degradation of mRNAs
The HIV genome, which contains several coding regions, is transcribed into a single 9-kb primary transcript. After transport to the cytoplasm, the various RNA species are translated into different viral proteins. Rev protein, encoded by a 2-kb mRNA, interacts with the Rev response element (RRE) in the un-spliced and singly spliced mRNAs, stimulating their transport to the cytoplasm. Processing of both miRNA precursors into miRNAs and long double-stranded RNAs into short interfering RNAs (siRNAs)
requires the Dicer ribonuclease. In miRNA function, the RISC RNA forms a hybrid with the target mRNA that contains some base-pair mismatches; in this case translation of the target mRNA is blocked. In immature oocytes, mRNAs containing the U-rich cytoplasmic polyadenylation element (CPE) have short poly(A) tails. Hormone stimulation of oocytes activates a protein kinase that phosphorylates CPEB, causing it to release Maskin. After the poly(A) tail is lengthened, multiple copies of the cytoplasmic poly(A)-binding protein I (PABPI) can bind to it and interact with eIF4G, which functions with other initiation factors to bind the 40S ribosome subunit and initiate translation. In the deadenylation-dependent pathways, the poly(A) tail is progressively shortened by a deadenylase until it reaches a length of 20 or fewer A residues at which the interaction with PABPI is destabilized.