Gene Expression Control by RNA W8

Control of Gene Expression by RNA

Introduction

Presenter: Dave Carter
Institution: Oxford Brookes University
Contact: dcarter@brookes.ac.uk
Main focus: This presentation delves into the intricate control of gene expression, specifically highlighting the diverse functions of various RNA types, with a particular emphasis on messenger RNA (mRNA) and microRNA (miRNA) beyond their traditional roles. Gene expression control involves the processes that turn genes on and off, ensuring specific proteins are made at the right time and in the right amounts.

Overview of Gene Count in Humans

Question: How many genes does it take to make a human? This question sets the stage for understanding genome complexity.
- Homo sapiens: The human genome contains approximately $20,000$ protein-coding genes.
- Caenorhabditis elegans (a model organism): This nematode worm, despite having far fewer cells (around $1,000$ somatic cells per organism) compared to the human body's over $100$ trillion cells, possesses a remarkably similar number of genes, also approximately $20,000$ .
- Comparison raises questions regarding the profound differences in organism complexity, physiology, and behavior despite similar gene counts. This suggests that complexity is not solely determined by the number of genes, but by how these genes are regulated, processed (e.g., alternative splicing), and the vast regulatory networks they form.

Number of Genes in the Human Genome

Estimated range: While the exact number is subject to ongoing refinement, current estimates place the human protein-coding gene count between $20,000$ to $25,000$ genes.
Notable mention: The striking similarity in gene numbers with much simpler organisms like worms underscores the importance of post-transcriptional and post-translational regulatory mechanisms in generating biological complexity.

Junk DNA Debate

Discussion on the historical concept and current understanding of 'junk' DNA:
- An increasing percentage of the human genome is now recognized as non-coding, initially raising questions about its functionality and leading some to label it 'junk'. This non-coding DNA includes introns, regulatory sequences (enhancers, promoters), pseudogenes, and repetitive elements.
Over 90% of the human genome, including significant portions of what was once considered 'junk' DNA, is transcribed into various forms of RNA, despite only approximately 2% of the genome being actual protein-coding genes. This observation strongly suggests that much of the non-coding RNA (ncRNA) must serve important biological functions.

The Central Dogma of Molecular Biology

Definition: The central dogma, formulated by Francis Crick, describes the fundamental flow of genetic information within a biological system: from DNA to RNA to protein. This process involves two main steps: transcription (DNA to RNA) and translation (RNA to protein).
RNA's primary role is to serve as a messenger (mRNA), conveying genetic sequences from the DNA in the nucleus to the ribosomes in the cytoplasm for translation into proteins.
Additionally, RNA performs various other essential functions, acting not just as a messenger but also as structural components (rRNA), transporters (tRNA), and crucial regulatory molecules (ncRNAs) that modulate gene expression at multiple levels.

The Secret Life of the Nucleus

Composition of the Genome:
- Approximately 2% of the human genome consists of protein-coding genes, which dictate the amino acid sequences of proteins.
- The remaining 98% is considered non-coding. Historically, this vast majority was often dismissed as 'junk' due to a lack of apparent coding capacity.
- However, recent genomic studies have revealed that a significant portion of this non-coding DNA is actively transcribed into various RNA molecules, leading to the classification of these transcripts as non-coding RNAs (ncRNAs).
The discussion poses a critical question: Do these ncRNAs serve any biological purpose, or are they merely transcriptional noise? Mounting evidence points towards their indispensable regulatory roles.

Roles of ncRNAs

Functions: ncRNAs have emerged as key players in cellular processes, executing a wide array of regulatory tasks:
- Regulation of gene transcription: They can influence the initiation and efficiency of transcription by interacting with transcription factors, RNA polymerase, or by directly modulating chromatin structure (e.g., recruiting chromatin-modifying enzymes).
- Regulation of protein production: This includes controlling mRNA stability, translation initiation, and translational efficiency, thereby impacting the quantity of protein produced from a given mRNA.
- Influence on nuclear structure and chromatin structure: ncRNAs can act as scaffolds, guiding proteins to specific genomic loci, or directly modulating the higher-order organization of chromatin. For instance, they can recruit Polycomb or Trithorax group proteins to modify histones, leading to gene repression or activation.

Evolution of Non-Coding RNAs

ncRNAs are observed to be evolving at a faster rate compared to protein-coding genes. This rapid evolution suggests that ncRNAs might be key drivers of evolutionary novelty and species-specific traits, allowing for quicker adaptation to environmental pressures.
Hypothesis: This accelerated evolution and their diverse regulatory capabilities suggest that ncRNAs could significantly enhance the complexity of organisms and their regulatory landscapes, even with a relatively limited number of protein-coding genes. They provide an additional layer of regulatory intricacy that can lead to diverse phenotypes.

Types of ncRNAs

Long ncRNAs

Sizes: Long ncRNAs (lncRNAs) are generally defined as RNA molecules greater than $200$ nucleotides (or sometimes $100$ base pairs) in length that do not encode proteins. This distinguishes them from short ncRNAs.
Roles: LncRNAs interact extensively with various proteins, including chromatin-modifying complexes, transcription factors, and RNA-binding proteins, to influence gene expression through a multitude of mechanisms.
Examples: Key examples include Xist, involved in X-chromosome inactivation; Air, implicated in genomic imprinting; Hotair, a regulator of HOXD gene expression; and lincRNA-p21, involved in cell cycle regulation.

Mechanisms of Long ncRNAs

X-chromosome Inactivation

Process ensuring equal expression of X-linked genes between male (XY) and female (XX) individuals. In female mammals, one of the two X chromosomes is largely silenced to achieve this dosage compensation.
- Xist function: X-inactive specific transcript (Xist) is a $17Kb$ long non-coding RNA. It is expressed from the inactive X chromosome and acts in cis to physically coat the entire chromosome. This coating is a critical initial step in X-chromosome inactivation, leading to the recruitment of inactivating proteins and the establishment of a heterochromatic state.
Recruits Polycomb Repressive Complex 2 (PRC2): Xist physically interacts with and recruits PRC2, a major epigenetic regulator. PRC2 then catalyzes specific histone modifications, such as methylation at lysine $27$ of histone H $3$ ( $ext{H3K27me3}$ ), which are hallmarks of transcriptional repression and help to establish and maintain chromatin inactivity across the entire X chromosome.

Genomic Imprinting

Imprinting: A fascinating epigenetic phenomenon where certain genes are expressed in a parent-of-origin specific manner; that is, only the allele inherited from either the mother or the father is expressed, while the other is silenced. Approximately $80$ genes in humans exhibit this phenomenon, playing crucial roles in development and disease.
Mechanism: Genomic imprinting generally involves differential DNA methylation patterns established during gametogenesis that specifically silence one parental allele. This epigenetic marking is often orchestrated and mediated by long ncRNAs, which can guide DNA methyltransferases or other chromatin modifiers to specific loci.

Example of Gene Imprinting with Air ncRNA

Air mRNA: The Air (antisense imprinted RNA) is a paternally expressed ncRNA. It plays a crucial role in regulating the expression of the imprinted gene IGF2R (Insulin-like Growth Factor 2 Receptor), which is maternally expressed and paternally repressed. Air is transcribed antisense to the Igt2r gene cluster.
Mechanism: The Air lncRNA recruits the histone methyltransferase G $9a$ to the IGF2R promoter region on the paternal allele. This recruitment leads to a series of epigenetic modifications, including methylation of lysine $9$ of histone H $3$ ( $ext{H3K9me2/3}$ ) and DNA methylation. These modifications ultimately establish a repressive chromatin state, leading to the silencing of IGF2R on the paternal chromosome.

HOTAIR and Gene Regulation

Role: HOTAIR (HOX Antisense Intergenic RNA) is a lncRNA transcribed from the HOXC locus. It acts in trans to repress the expression of HOXD cluster genes, which are critical for embryonic development and body patterning. Similar to Xist and Air, HOTAIR achieves this by recruiting chromatin-modifying complexes, particularly PRC2, to the HOXD locus.
Conservation: A remarkable aspect of lncRNAs like HOTAIR is that while their primary nucleotide sequence diversity can be high across species, their structural features (e.g., secondary or tertiary RNA structures) and their functional mechanisms (e.g., interacting with PRC2) may remain highly conserved. This structural conservation is crucial for their function and provides insights into their evolutionary stability despite sequence divergence.

Short ncRNAs

Description and Examples

Types: This category includes small interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), and microRNAs (miRNAs). Each type has distinct biogenesis pathways and targets specific gene regulatory processes.
tRNA and rRNA are also types of ncRNAs, but their primary roles are structural (rRNA, which forms the core of ribosomes) and adapter (tRNA, which carries amino acids during translation). They are not primarily involved in dynamic gene regulation in the same way as siRNAs, piRNAs, and miRNAs, which actively modulate gene expression.

MicroRNAs (miRNAs)

Definition: miRNAs are small, non-coding RNA molecules, typically single-stranded and approximately $20-23$ nucleotides in length. They play a critical role in post-transcriptional gene regulation.
Discovery History: The first miRNA, lin-4, was discovered in C. elegans in $1993$ , found to regulate the lin-14 gene. Another seminal discovery was let-7 in $2000$ , also in C. elegans, which was later found to be evolutionarily conserved across many species, including humans, underscoring the ancient and fundamental nature of miRNA regulation.
Roles: Since their initial discovery, thousands of miRNAs have been identified across various organisms, demonstrating their ubiquitous involvement in diverse biological processes, including development, cell proliferation, differentiation, apoptosis, and metabolism. They exert their influence primarily by regulating gene expression at the post-transcriptional level.

MicroRNA Biogenesis

miRNAs are produced through a multi-step enzymatic pathway. They are initially transcribed as long primary transcripts (pri-miRNAs), which are hundreds or thousands of nucleotides long and fold into distinctive hairpin structures.
These pri-miRNAs are then processed in the nucleus by the Microprocessor complex, consisting of the Drosha enzyme (a ribonuclease III) and its cofactor DGCR $8$ , into shorter precursor miRNAs (pre-miRNAs), typically around $70$ nucleotides, still retaining a hairpin structure. These pre-miRNAs are then exported to the cytoplasm, where they are further processed by another ribonuclease III enzyme, Dicer, into a mature double-stranded miRNA duplex (approximately $20-23$ bp).
Functional importance of double-stranded RNA: The involvement of double-stranded RNA in miRNA biogenesis is crucial, linking it to broader RNA interference (RNAi) mechanisms. RNAi pathways are essential for slowing down the replication of certain viruses and for precise regulation of gene expression, often by degrading target mRNAs or inhibiting their translation.

MicroRNA Function in Gene Regulation

Mechanism: After maturation, one strand of the miRNA duplex is loaded into the RNA-induced silencing complex (RISC). The miRNA within RISC then guides the complex to specific target messenger RNAs (mRNAs) based on sequence complementarity, primarily within a region called the 'seed region'. This binding can lead to either the degradation of target mRNAs (if complementarity is extensive) or the inhibition of their translation (if complementarity is imperfect).
Seed Region: This is a critical $6-8$ nucleotide sequence, typically at positions $2-7$ from the miRNA's $5'$ end. The seed region's specific sequence defines the miRNA's target mRNAs, enabling a single miRNA to regulate a multitude of different genes simultaneously, thereby coordinating complex cellular responses.
Endogenous role: miRNAs function as naturally occurring, endogenous regulators of gene expression, distinct from exogenous siRNAs that are typically introduced experimentally or arise from viral infection. While both utilize the RISC complex, miRNAs primarily fine-tune gene expression by repressing translation or degrading mRNA targets.

Examples and Impacts of miRNA in Cancer

Oncogenic miRNAs like Mir- $21$ : Certain miRNAs are frequently dysregulated in cancer. For instance, Mir- $21$ is often upregulated in various human cancers (e.g., breast, colon, lung cancer). It acts as an oncomiR by repressing the expression of tumor suppressor genes such as PTEN (Phosphatase and Tensin Homolog) and PDCD4 (Programmed Cell Death $4$ ). By reducing the activity of these tumor suppressors, Mir- $21$ promotes cell proliferation, inhibits apoptosis, and enhances tumor growth and metastasis.
Highlighted functions: Beyond cancer, miRNAs also impact important clinical and biological processes such as cell differentiation (e.g., specific miRNAs can be manipulated to convert fibroblasts into induced pluripotent stem cells or even directly to neurons), development, and immune responses.

Competing Endogenous RNA (ceRNA) Hypothesis

Concept: The ceRNA hypothesis proposes a novel regulatory layer where various RNA transcripts—including pseudogenes, long non-coding RNAs (lncRNAs), and circular RNAs—can communicate with each other by competitively binding to shared miRNAs. These RNAs act as 'decoys' or 'sponges' for miRNAs, thereby altering the effective concentration of free miRNAs available to regulate their bona fide coding mRNA targets.
Notable study: An exemplary interaction demonstrating this decoy function is observed between the PTEN tumor suppressor gene and its pseudogene, PTENP1. PTENP1 shares sequence similarity with PTEN and thus possesses common miRNA binding sites. When PTENP1 transcript levels are high, it sponges miRNAs that would otherwise target PTEN mRNA, leading to increased PTEN expression. This emphasizes a novel, extensive regulatory network beyond simple coding capacity, where non-coding RNAs significantly modulate the levels of protein-coding genes.

Conclusion

The cumulative evidence emphatically emphasizes the profound importance of RNA beyond its mere coding functions. This presentation showcases its vast and intricate regulatory capabilities in controlling virtually every aspect of gene expression and a myriad of cellular processes, highlighting RNA as a central conductor of biological complexity.