RNA Processing and Gene Structure Study Notes

RNA Molecules and RNA Processing

Introduction

  • Overview of important concepts in RNA biology and processing, focusing on pre-mRNA modifications.

Key Concepts in Gene Structure

  • Genes Have Complex Structures

    • In bacteria and viruses, there is a colinear relationship between the nucleotide sequence of a gene and the amino acid sequence of the corresponding protein.

    • In eukaryotes, such as humans, this colinear relationship does not exist.

    • Eukaryotic genes, and some prokaryotic genes, are often longer than the mRNA they encode.

    • Introns (noncoding regions) are present within these longer genes, interrupting the coding regions, known as exons.

    • The RNA transcribed by RNA polymerase from the gene, which includes both introns and exons, is referred to as the primary transcript (pre-mRNA).

RNA Processing

  • Processing Overview

    • After transcription, pre-mRNA undergoes several modifications before becoming mature mRNA.

    • These modifications include:

    • Capping

    • Polyadenylation

    • Splicing

  • Structure of mRNA

    • Primary transcript includes the entire length of RNA with introns and exons.

    • In eukaryotes, mRNA must be processed.

    • RNA does not require post-transcriptional modifications in most bacteria as mRNA is immediately translatable.

  • Each amino acid in a protein is signified by a triplet of nucleotides called a codon. Additionally, important elements in the mRNA structure include:

    • Ribosome binding site: Just upstream of the start codon.

    • Untranslated regions (UTRs): These are sequences at both 5’ and 3’ ends essential for stability and initiation of translation.

Pre-mRNA Processing Events

  • There's a typical sequence of processing events that pre-mRNA undergoes:

    1. Addition of 5' Cap

    2. Addition of Poly(A) Tail

    3. Splicing

Addition of 5' Cap

  • The 5' cap addition involves:

    • Addition of 7-methyl guanine to the 5’ end of pre-mRNA.

    • This cap includes methylation of the 2’ OH group of one or two ribose sugar nucleotides near the 5’ end of pre-mRNA.

    • Capping enzymes attach at the outlet of the exit channel of RNA polymerase to facilitate this process.

    • Functions of the 5' Cap:

    • Increases mRNA stability by protecting against degradation by exonucleases, which attack the exposed ends of mRNA.

    • Binding by cap-binding proteins facilitates ribosome attachment and initiation of translation.

Addition of Poly(A) Tail

  • Polyadenylation refers to the addition of a poly(A) tail, consisting of 50 to 250 adenine nucleotides at the 3’ end of pre-mRNA.

    • Functions of the poly(A) tail include:

    • Enhancing mRNA stability, which is influenced by the tail’s length.

    • Initiating translation, as the tail aids in ribosome attachment after circularization of the mRNA.

    • Facilitating export of the mRNA to the cytoplasm.

RNA Splicing

  • Splicing Mechanism

    • Involves the removal of introns and joining of exons.

    • Occurs within the nucleus before mRNA is exported.

    • Three critical sequences in an intron are essential for proper splicing. Mutations disrupting these can lead to disease.

    • The branch point is located 18-46 nucleotides upstream from the 3’ splice site, with no consensus sequence in the branch point region.

  • Spliceosome Composition

    • The spliceosome is assembled from five small nuclear ribonucleoprotein particles (snRNPs) and additional proteins, with snRNPs containing snRNA and proteins.

    • Certain snRNPs are responsible for recognizing splice site junctions and branch points.

Alternative Processing Pathways

  • A single pre-mRNA can undergo different processing routes, leading to alternative mRNA forms, which results in the production of various proteins from the same gene.

    • This challenges the traditional view of a gene being just a straightforward sequence of nucleotides coding for a specific protein sequence.

    • Examples of Alternative Processing:

    • Alternative Splicing: Different combinations of exon splicing.

    • Multiple 3’ Cleavage Sites: Presence of various cleavage points in pre-mRNA.

    • Example: Pre-mRNA processing for the calcitonin gene can produce calcitonin in thyroid cells and calcitonin gene-related peptide (CGRP) in brain cells.

RNA Editing

  • RNA editing modifies the coding sequence of mRNA post-transcription, resulting in potential changes in the protein's amino acid sequence.

  • Mechanisms of RNA Editing:

    1. Using guide RNAs (gRNAs) that contain sequences partially complementary to the target pre-edited RNA to direct modifications.

    2. Base conversion, as seen in the alternative processing of apolipoprotein B mRNA, where C is converted to U at a specific codon, producing a stop codon leading to truncated protein synthesis.

RNA Interference (RNAi)

  • Mechanism Overview:

    • RNA interference is a gene silencing method utilized by eukaryotic cells involving small RNA molecules, typically 21-25 nucleotides long.

    • Serves a protective role against RNA viruses.

    • Triggered by double-stranded RNA molecules that may produce:

    • Small Interfering RNAs (siRNAs): Derived from double-stranded RNA, targeting mRNA for degradation with exact complementarity.

    • Micro RNAs (miRNAs): Can inhibit translation and may derive from distinct miRNA genes or from mRNA exons/introns, forming hairpin structures.

Summary of Learning Outcomes

  • Students should achieve the following:

    • Understand and explain new terminology relevant to RNA processing and editing.

    • Describe the various forms of RNA processing and their significance.

    • Analyze how alternative processing pathways can influence gene expression in a tissue-specific manner.

    • Distinguish between different types of RNA editing.

    • Assess the mechanisms by which miRNAs and siRNAs operate in gene regulation and expression.