The Expression of Genetic Information via Genes II: Non-coding RNAs

Chapter 11 – The Expression of Genetic Information via Genes II: Non-coding RNAs

Chapter Outline

  • 1. Overview of non-coding RNAs

  • 2. Role of ncRNAs in eukaryotic DNA replication

  • 3. Effects of ncRNAs on chromatin structure and transcription

  • 4. Effects of ncRNAs on translation and mRNA degradation

  • 5. ncRNAs and protein sorting

  • 6. ncRNAs and genome defense

  • 7. Roles of ncRNAs in human disease and plant health

11.1 Overview of Non-coding RNAs

Section 11.1 Learning Outcomes

  • Describe the ability of ncRNAs to bind to other molecules and macromolecules.

  • Outline the general functions of ncRNAs.

  • Define ribozyme.

  • List several examples of ncRNAs, and describe their functions.

Overview of Non-coding RNAs

  • Experimental tools to study RNA structure and function are more recently developed than those for proteins and protein-encoding genes.

  • The human genome contains approximately 22,000 protein-encoding genes.

  • Non-coding RNAs (ncRNAs) are RNA molecules that do not encode polypeptides but are transcribed from other genes.

  • In most cell types, ncRNAs are more abundant than mRNAs, with only 20% of transcription typically leading to mRNA production in human cells.

  • Historical bias exists against RNA, as most biology education has focused on DNA and protein functions; this chapter aims to explore the structure, function, and biological roles of ncRNAs.

ncRNAs Binding Capability

  • ncRNAs can bind to other RNA or DNA through complementary base pairing. This enables them to influence critical processes such as:   - DNA replication   - Transcription   - Translation

  • They can also interact with proteins or small molecules, utilizing stem-loop structures to bind or scaffold proteins or create binding sites for small molecules.

Functions of ncRNAs

  • Common functions of ncRNAs include:   - Scaffold: Bind to multiple components (e.g., proteins) to facilitate the formation of a complex.   - Guide: Direct one molecule to a specific cellular location, such as guiding a protein to a site on DNA.   - Alteration of Protein Function or Stability: Binding of ncRNAs can alter a protein's catalytic activity, its capacity to bind other molecules, or its overall stability.

  • Ribozyme: An RNA molecule with catalytic properties, exemplified by the peptidyltransferase activity of ribosomes.

  • Blocker: ncRNA that prevents a cellular process from occurring; for instance, by obstructing the binding of a ribosome and thereby inhibiting translation.

  • Decoy: Sequesters other ncRNAs to inhibit their function. The distinction between a blocker and decoy is rooted in the type of molecules they bind to.

Examples of ncRNAs

Type of ncRNA

Plays a Role in

Description

Telomerase RNA component (TERC)

DNA replication

Facilitates telomerase binding to the telomere and serves as a template for DNA replication.

X inactive specific transcript (Xist RNA)

Chromatin structure, transcription

Coats one X chromosome in female mammals, aiding in its compaction and subsequent inactivation.

Hox transcript antisense intergenic RNA (HOTAIR)

Chromatin structure, transcription

Alters chromatin structure to silence genes by guiding histone-modifying complexes.

Transfer RNA (tRNA)

Translation

Recognizes mRNA codons and carries specific amino acids accordingly.

Ribosomal RNA (rRNA)

Translation

Forms part of ribosomes, the sites for polypeptide synthesis.

microRNA (miRNA), small-interfering RNA (siRNA)

Translation, RNA degradation

Regulate mRNA expression and degradation.

RNA component of signal recognition particle (SRP-RNA)

Protein sorting, secretion

Directs polypeptide synthesis to the plasma membrane in bacteria and to the endoplasmic reticulum in eukaryotes.

CRISPR RNA (crRNA)

Genome defense

Guides a nuclease to foreign DNA, such as bacteriophage DNA, in bacteria and archaea.

11.2 Role of ncRNAs in Eukaryotic DNA Replication

Section 11.2 Learning Outcomes

  • Describe the structure and function of telomeres.

  • Explain how the ncRNA known as TERC plays a role in the replication of telomeres in eukaryotes.

Telomeres in Eukaryotic DNA Replication

  • Linear eukaryotic chromosomes' ends consist of telomeres made of repetitive sequences, particularly 5ʹ–GGGTTA–3ʹ in humans, repeating hundreds of times.

  • Telomeres safeguard chromosome ends from tangling or breaking. Shortening occurs during cell division, leading to programmed cell death when they become critically short.

  • Cells that proliferate rapidly express telomerase, an enzyme that extends telomeres by adding repeating sequences.

TERC and Telomere Replication

  • The capability of DNA polymerase is limited, as it can only synthesize in the 5ʹ to 3ʹ direction and requires a pre-existing strand for extension.

  • This constraint results in the inability to fully replicate the 3ʹ ends, resulting in a 3ʹ overhang post-replication.

  • Telomerase, consisting of proteins and an ncRNA called TERC (telomerase RNA component), prevents chromosome shortening through three steps:   1. Binding of Telomerase: TERC guides telomerase to the complementary DNA repeat.   2. Polymerization: TERC provides a template for synthesizing a six-nucleotide sequence, facilitated by the reverse transcriptase activity of telomerase.   3. Translocation: Telomerase shifts to the newly synthesized DNA end and continues the addition of further nucleotides.

11.3 Effects of ncRNAs on Chromatin Structure and Transcription

Section 11.3 Learning Outcomes

  • Explain how the ncRNA known as HOTAIR plays a role in regulating gene expression.

HOTAIR Function in Gene Regulation

  • HOTAIR (Hox transcript antisense intergenic RNA) regulates transcription by acting as a scaffold to bind two protein complexes, guiding them to specific genes.

  • These protein complexes chemically modify histones, leading to gene silencing through chromatin modifications.

11.4 Effects of ncRNAs on Translation and mRNA Degradation

Section 11.4 Learning Outcomes

  • Analyze experimental evidence that double-stranded RNA is more potent at inhibiting mRNA than is antisense RNA.

  • Compare and contrast microRNAs (miRNAs) and small-interfering RNAs (siRNAs).

  • Outline the steps of RNA interference and detail the events occurring during each step.

Potency of Double-Stranded RNA

  • ncRNAs can modulate mRNA translation and degradation.

  • The experimentation conducted by Fire and Mello aimed to discern the impact of RNA injection on silencing specific mRNAs, specifically studying the mex-3 gene in C. elegans.   - Embryos received injections of either single-stranded RNA complementary to mex-3 mRNA or double-stranded RNA.   - Results indicated that embryos with single-stranded RNA showed reduced mex-3 mRNA levels, but those with double-stranded RNA had undetectable mex-3 mRNA, confirming that the latter leads to mRNA degradation, a mechanism known as RNA interference (RNAi).

Mechanisms of RNA Interference

  • RNA interference is a process found across eukaryotic species that involves two sources of ncRNA:   1. MicroRNAs (miRNAs): Derived from endogenous eukaryotic genes and regulate gene expression, with partial complementarity to target mRNAs. Approximately 60% of human protein-coding genes are affected by miRNAs.   2. Small-interfering RNAs (siRNAs): Typically originate from exogenous sources such as viral infections or experimental manipulation. siRNAs are usually perfectly complementary to their mRNA targets and are crucial in antiviral defense and research applications.

  • In the nucleus, miRNAs are synthesized as primary-miRNAs and fold into hairpin structures. Cleavage by Dicer enzyme produces 20 to 25 bp double-stranded miRNAs and siRNAs in the cytosol.

  • The double-stranded RNA then associates with proteins to form the RNA-induced silencing complex (RISC), with one strand degraded while the other guides the complex to target mRNAs. The bound mRNA is then inhibited from translation or subjected to degradation.

11.5 Non-coding RNAs and Protein Sorting

Section 11.5 Learning Outcomes

  • Describe the structure and function of the signal recognition particle (SRP).

  • Explain the roles of SRP RNA concerning SRP function.

Role of SRP in Protein Targeting

  • Proteins must be directed to specific locations for functional execution, facilitated by the RNA-protein complex known as the signal recognition particle (SRP).

  • In bacteria, SRP comprises one ncRNA and one protein; in eukaryotes, it consists of one ncRNA and six proteins.

  • The SRP RNA has two primary functions:   1. Provides a scaffold for protein binding.   2. After binding to the SRP receptor in the ER membrane, it stimulates GTP hydrolysis by proteins within the SRP and SRP receptor, altering their structure to enhance GTPase activity.

Steps in Polypeptide Targeting via SRP

  1. During polypeptide synthesis, SRP binds to an ER signal sequence leading to a pause in translation.

  2. SRP then associates with its receptor in the ER membrane, which is adjacent to a protein channel; GTP-binding proteins are also required for this interaction.

  3. Hydrolysis of GTP by these proteins results in the release of the SRP, allowing translation to resume and threading the polypeptide into the ER lumen through the channel.

11.6 Non-coding RNAs and Genome Defense

Section 11.6 Learning Outcomes

  • Outline the genes that are part of the type II CRISPR-Cas system.

  • Explain how the CRISPR-Cas system defends bacteria against bacteriophages.

  • Explain the adaptation, expression, and interference stages of the CRISPR-Cas system.

CRISPR-Cas System Overview

  • Some bacterial and archaeal species utilize the CRISPR-Cas system for defense against bacteriophages and transposable elements, classified into three groups: I, II, and III. The type II CRISPR-Cas system specifically equips bacteria against bacteriophage threats.

Components of the Type II CRISPR-Cas System

  • The system includes:   - Crispr Gene: Features repeated sequences interspersed with unique spacer sequences.   - tracrRNA (trans-activating CRISPR RNA): An ncRNA critical for CRISPR function.   - Cas Genes: Encodes several CRISPR-associated proteins.

  • The defense mechanism unfolds in three main stages: adaptation, expression, and interference.

Phases of the CRISPR-Cas System

  1. Adaptation: Following exposure to bacteriophage DNA, the Cas1 and Cas2 proteins recognize and cleave the foreign DNA, incorporating it into the Crispr gene.

  2. Expression: In previously adapted bacteria, exposure to the bacteriophage triggers gene transcription, producing tracrRNA and crRNA molecules. The tracrRNA has complementary regions to the repeats of pre-crRNA, enabling processing into small crRNA units.

  3. Interference: The assemblage of tracrRNA and crRNA with Cas9 allows for the recognition of foreign DNA during bacteriophage infections and cleavage of the phage DNA, reminiscent of RNA interference processes.

11.7 Role of ncRNAs in Human Disease and Plant Health

Section 11.7 Learning Outcomes

  • List examples where ncRNAs are associated with human diseases.

  • List examples of the roles ncRNAs play in plant health.

ncRNAs and Human Diseases

  • Abnormal levels of certain miRNAs correlate with various human cancers, where some act as tumor suppressors while others behave as oncogenes.

  • HOTAIR, known to be overexpressed in many cancers, functions as an oncogene of note.

  • Various ncRNAs are linked to neurological disorders (e.g., Alzheimer's disease) and cardiovascular diseases (e.g., arrhythmias).

ncRNAs and Plant Health

  • Plant research highlights the fundamental roles of ncRNAs in crucial processes such as seed development, growth, and responses to environmental stress, emphasizing their significance in agriculture.