Molecular Biology Chapter 14: RNA Processing-I: Splicing

Post-transcriptional Processing in Eukaryotes - Genetic Interpretation: Most eukaryotic genes, in contrast to typical bacterial genes, are interrupted by noncoding DNA. - Transcription Realities: RNA polymerases cannot distinguish the noncoding regions from the coding regions, resulting in the transcription of the entire gene sequence. - Processing Requirements: The cell must perform splicing to remove noncoding RNA from the primary transcript. - Supplemental Structures: Eukaryotes add a 5' cap and a 3' poly-A tail to the transcript. - Cellular Localization: All processing events occur within the nucleus before the mature mRNA emigrates to the cytoplasm. # Evidence for Split Genes - Defining Components: - Introns: These are non-coding, intervening sequences that interrupt coding regions. - Exons: These contain the sequences that finally appear in the mature RNA product. - Statistical Variation: - Genes for mRNAs have been identified with a range from $0$ to $362$ introns. - tRNA genes contain either $0$ or $1$ intron. # RNA Splicing Mechanics - Fundamental Process: RNA splicing is the specific process of cutting introns out of immature RNAs and stitching together the exons to form the final product. - Sequential Stages of Splicing: - 1st Stage (Synthesis of Primary Transcript / Pre-mRNA): This is the initial RNA molecule synthesized by RNA polymerase II from a DNA template. It is a direct copy of the gene, containing both exons and introns. - 2nd Stage (mRNA Maturation): The actual removal of introns through the splicing mechanism. # Splicing Signals and Conserved Motifs - 5′ Splice Site (Donor): The intron begins with a highly conserved $GU$ sequence, which is recognized by the U1 snRNP. - 3′ Splice Site (Acceptor): The intron ends with a highly conserved $AG$ sequence, which is crucial for recognition by U2AF. - Consensus Sequences: Proper splicing depends on whole consensus sequences extending beyond the $GU$ and $AG$ motifs. # Two-Step Model of Splicing Mechanism - First Step: The $2'-OH$ group of an adenosine nucleotide located in the middle of the intron attacks the phosphodiester bond between the 1st exon and the $G$ at the beginning of the intron. This forms the loop of the lariat and separates the first exon from the intron. - Second Step: The $3'-OH$ left at the end of the 1st exon attacks the phosphodiester bond linking the intron to the 2nd exon. This forms the exon-exon phosphodiester bond and simultaneously releases the intron in lariat form. # Signals at the Branch - Importance: Along with the 5' and 3' consensus sequences, crucial branchpoint consensus sequences occur. - Yeast Organisms: Sequence is invariant: $UACUAAC$. - Higher Eukaryotes: The consensus sequence is more variable than in yeast. # Spliceosomes and snRNPs - Definition: A large, dynamic ribonucleoprotein (RNP) complex in the eukaryotic nucleus that catalyzes pre-mRNA splicing. - Structural Components: - Pre-mRNA. - SnRNPs (“snurps”). - Splicing protein factors. - The snRNPs Specifics: - Consist of small nuclear RNAs (snRNAs) coupled to proteins. - Resolution: snRNAs can be resolved on a gel as $U1$, $U2$, $U4$, $U5$, and $U6$. - Commonality: All snRNPs share the same set of 7 Sm proteins. - Specificity: Each snRNP also possesses its own set of unique proteins. # The Spliceosome Cycle: Assembly and Function - Regulation: By controlling assembly, a cell regulates the quality and quantity of splicing, thereby regulating gene expression. - Stepwise Assembly: - 1. Integration of U1: U1 binds to the 5′ splicing site, forming a commitment complex. - 2. Integration of U2: U2 joins the complex to form the ‘A complex’; this requires $ATP$. - 3. Integration of U4-U6 and U5: These join to form the ‘B1 complex’. - Activation and Splicing: - U4 dissociates from U6 to allow U6 to displace U1 at the 5′-splice site in an $ATP$-dependent manner, activating the spliceosome. - U1 and U4 exit the complex. - U6 base pairs with U2 to form the ‘B2 complex’. - $ATP$ provides energy for the first splicing step, forming the ‘C1 complex’. - Another molecule of $ATP$ facilitates the second splicing step, forming the ‘C2 complex’. # Alternative Splicing - Frequency: Occurs in more than $50$% of human genes. - Patterns: Many genes have more than 2 patterns; some have thousands. - Biological Consequences: - Differentiation between secreted or membrane-bound proteins. - Determination of protein activity and inactivity. # Self-splicing RNAs - Overview: Some RNAs catalyze their own splicing without aid from a spliceosome or proteins. - Group I Introns: - Occurrence: Found in all life domains. - Bacterial context: Primarily in rRNA and tRNA genes. - Organelle context: Found in rRNA, tRNA, and protein-coding genes of fungal mitochondria, plant mitochondria, and chloroplasts. - Mechanism: Begins with an attack by a free guanine nucleotide on the 5′-splice site. The $G$ is added to the 5′-end of the intron. The first exon is released and then attacks the 3′-splice site to ligate exons and release a linear intron. - Group II Introns: - Occurrence: Found in organelle rRNA, tRNA, and mRNA (fungal/plant mitochondria, chloroplasts) and bacterial mRNA. - Mechanism: Involves an intramolecular attack by an $A$ residue within the intron to form a lariat structure.