RNA Diversity and Roles in Gene Expression (Lecture 3)

RNA Intro

• RNA stands for Ribonucleic Acid

• It is a macromolecule and a polymer composed of covalently linked monomers

• RNA monomers are nucleotides (ribonucleotides)

• Nucleotide chemistry referenced as: nucleotides linked by phosphodiester bonds

• RNA sugar is ribose (as opposed to deoxyribose in DNA)

• RNA contains uracil (U) instead of thymine (T)

• RNA is typically single-stranded

RNA: Basic Components and Properties

• Four RNA nucleotides: PURINES - Adenine (A) and Guanine (G); PYRIMIDINES - Cytosine (C) and Uracil (U)

• Diversity of RNA sequences arises from the order of these monomers

• RNA is usually single-stranded, but can fold into 3D structures via complementary base pairing

• Single-stranded RNA can fold on itself to form local 3D structures stabilized by short regions of base pairing

Transcription: The DNA-to-RNA Synthesis

• Transcription is the DNA-directed synthesis of RNA

• All eukaryotic cells contain 5 major classes of transcribed RNA:
  $\text{rRNA}, \quad \text{mRNA}, \quad \text{tRNA}, \quad \text{snRNA}, \quad \text{miRNA}$

• In mammals, 3 distinct RNA polymerases transcribe different RNA species:

  • Pol I: Synthesis of ribosomal RNA (rRNA)

  • Pol II: Synthesis of messenger RNA (mRNA)

  • Pol III: Synthesis of transfer RNA (tRNA), some rRNA, and small nuclear RNA (snRNA)

Roles of RNA in Gene Expression (Overview)

• Information in DNA directs the synthesis of proteins via transcription and translation

• Proteins carry out cellular functions (building structures, acting as enzymes, etc.)

• The 3 main RNA types directly involved in protein synthesis:
  $\text{mRNA}, \quad \text{rRNA}, \quad \text{tRNA}$

Messenger RNA (mRNA) and Translation

• mRNA carries the genetic message from DNA to the protein synthesis machinery

• When a protein is required, its coding gene is 'switched on'

• mRNA is synthesized by transcription

• mRNA interacts with the ribosome and tRNA to synthesize proteins during translation

• The ribosome is composed of rRNA and proteins (up to 60% of ribosome mass)

• rRNA guides mRNA binding for translation and ensures correct alignment of mRNA, tRNA, and ribosome; rRNA also has peptidyl transferase enzymatic activity (catalyzes peptide bond formation between amino acids)

The Ribosome and rRNA

• Ribosomes are the site of translation (peptide synthesis and translation of the genetic code)

• Eukaryotic ribosome is 80S, composed of two subunits: 60S and 40S

• The subunits assemble into a functional 80S ribosome when bound to mRNA

• rRNA is the most abundant cellular RNA in eukaryotes

• An 80S ribosome contains 4 distinct rRNA molecules and about 79 proteins

• The three largest rRNA molecules are 28S, 18S, and 5.8S

Transfer RNA (tRNA) – The Translator

• tRNA is the third main RNA type; one of the smallest (≈70–90 nt)

• Carries the next amino acid to the ribosome for protein synthesis

• Anticodon on tRNA base-pairs with the corresponding mRNA codon to ensure correct amino acid incorporation

• tRNA structures: 2D and 3D conformations exist

• There are about 45 types of tRNA, each linking a specific mRNA codon to its amino acid

• Some tRNAs recognize more than one codon (wobble at the 3′ base of the codon)

• tRNAs are recycled through multiple rounds of amino acid charging and delivery to the ribosome

RNA Structure and Function: Main Cellular RNA Types

• Three main RNA classes exist in all prokaryotic and eukaryotic organisms:

  • mRNA, tRNA, rRNA

• Other classes include: hnRNA, SnoRNA, miRNA, siRNA, SnRNA

Non-Coding RNAs (ncRNA) and Regulation

• ncRNA = RNA molecules not translated into proteins; umbrella term

• Regulation of genome organization and gene expression is increasingly elaborate in complex organisms; many post-transcriptional regulation mechanisms depend on ncRNAs

• The first regulatory RNAs discovered were microRNAs (miRNAs) in 1993 (regulate timing of C. elegans development)

• ncRNAs regulate physiological, developmental, and disease processes; most protein-coding genes are targets for miRNA-mediated regulation; miRNA function depends on sequence complementarity between miRNA and mRNA

• About ~1000 miRNA genes identified; ~2500 mature miRNAs identified

• miRNA regulation is a highly conserved mechanism

MicroRNAs (miRNAs): Biogenesis and Function

• miRNA genes are transcribed by RNA Pol II

• miRNA genes can occur within introns of protein-coding genes

• Mature miRNAs are short single-stranded RNAs (≈21–25 nt)

• miRNA biogenesis involves Dicer processing and incorporation into Argonaute-containing complexes

• RNA-induced silencing complex (RISC) is the minimal complex for regulation; mature miRNA bound to Argonaute constitutes RISC

• The miRNA guides RISC to mRNA targets by base-pairing with complementary sequences in the target (miRNA target sites) – commonly in the 3' UTR of mRNA

• Binding does not require perfect complementarity; the seed sequence (nt 2–8) is the minimal requirement for binding

• Mechanisms of RISC-mediated regulation include translational repression (initiation and elongation blocks), deadenylation and decapping leading to degradation, and sometimes mRNA cleavage

• Each miRNA sequence is unique; a single miRNA can regulate many mRNA targets; one mRNA can have multiple miRNA binding sites

• RISC acts to fine-tune gene expression and regulates the majority of human genes post-transcriptionally, enabling rapid responses to environmental changes

• Altered miRNA expression is linked to disease; cancer tissues often show global decreases in miRNA expression; some miRNAs act as tumor suppressors, while others (oncomirs) are overexpressed in cancers

Long Non-Coding RNAs (lncRNAs)

• Long non-coding RNAs (lncRNAs): >200 nt; single-stranded RNAs transcribed by RNA Pol II; undergo processing similar to mRNA

• The human genome encodes thousands of lncRNAs with diverse functions

• Roles include regulation of gene expression at transcriptional and post-transcriptional levels; they can act as decoys or sponges for miRNAs (ceRNA mechanism) by sharing sequence similarity with mRNA

• lncRNAs may recruit transcription factors to promoters or modulate chromatin states (epigenetic regulation)

• Example: PTEN and its pseudogene lncRNA PTENP1 act as miRNA sponges; PTENP1 competes with PTEN for miRNA binding, thereby freeing PTEN translation when PTENP1 miRNA binding reduces repression

• PTEN is a tumor suppressor; PTENP1 pseudogene-derived lncRNA can modulate PTEN levels through miRNA interactions

ncRNA: Summary and Significance

• The genome encodes many classes of ncRNA with diverse size, biogenesis, conservation, and function

• ncRNAs help explain biological questions about evolution, disease, and genomic complexity

RNA as the Genetic Material (RNA Viruses)

• RNA can serve as the genetic material for some viruses when they lack DNA

• Examples of RNA viruses: Rhinoviruses (common cold), influenza viruses, Ebola virus

• Overview of viral life cycle (illustrated in slides): transcription, splicing, translation, RNA replication, and assembly

Transcriptome: The World of RNA Transcripts

• The transcriptome = the complete collection of RNAs transcribed in the cell

• Not all transcribed RNAs code for protein; only mRNA codes for proteins

• Approximately 90% of the human genome is transcribed, but only about 3% of transcribed RNA codes for proteins

• In contrast, bacteria such as E. coli have ~4000 genes in a ~5 million bp genome with ~90% coding for protein; C. elegans has ~21,700 genes in ~100 million bp with ~25% coding for protein

Pre-mRNA Processing: From hnRNA to Mature mRNA

• Transcription produces a primary transcript (pre-mRNA or hnRNA, heterogeneous nuclear RNA)

• Pre-mRNA processing occurs in the nucleus to form mature mRNA

• 5' capping: A 5' cap is a modified guanine (meG) added during co-transcriptional processing; functions include protection from degradation, export from nucleus, and essential for ribosome attachment

• 3' Poly(A) tail: Addition of 20–250 adenines (A) at the 3' end; tail protects from degradation, facilitates nuclear export, and is bound by Poly(A) Binding Protein (PABP); essential for translation

• Intron/spliceosome: Genes often contain introns (non-coding) interspersed with exons (coding); introns are spliced out and exons joined to form mature mRNA

• Average eukaryotic transcription unit length ~8 kb; coding sequence is not continuous in DNA

• Splicing occurs via the spliceosome, a complex of snRNPs composed of protein and small nuclear RNA (snRNA); snRNPs recognize intron sequences and catalyze splicing

• Splice sites: 5' splice site = GU; 3' splice site = AG; Branch Point Sequence (BPS) important for lariat formation

• snRNA within the spliceosome plays a catalytic role (ribozyme activity)

• Alternative splicing can generate multiple polypeptides from a single gene by joining different exons/introns combinations

• About ~98% of human genes are alternatively spliced; key mechanism for protein diversity in higher eukaryotes; crucial for tissue-specific expression

• Example: Human tropomyosin gene shows multiple exons; different tissues show different splicing patterns leading to tissue-specific protein isoforms

• Regulation of splicing is complex and highly regulated; signals encoded in pre-mRNA sequence; many splicing events are cell-, tissue-, or environment-dependent

• Approximately 15% of disease-causing mutations affect splicing

Mature mRNA: Structure and Translation Signals

• Coding Sequence (CDS) is translated into a polypeptide

• Start codon is AUG (codes for methionine, M)

• Stop codons are UAG, UAA, or UGA and signal termination of translation (these do not code for amino acids)

• 5'-untranslated region (5'-UTR): Recognized by ribosome to allow translation initiation

• 3'-untranslated region (3'-UTR): Contains regulatory sequences for translation and post-transcriptional control; also involved in termination and modification

• Overall, mRNA acts as the ultimate information carrier from DNA to ribosomal protein synthesis machinery

• mRNA is single-stranded and has variable length depending on gene and protein size

Central Dogma Context

• The central dogma frames the flow of genetic information: DNA -> RNA -> Protein

• mRNA serves as the messenger from the DNA-encoded information to the protein-synthesizing machinery

Part 2: Regulatory Roles of RNA (Non-Coding RNAs) and miRNA

Overview of Part 2

• Focus on regulatory roles of RNA, especially non-coding RNAs (ncRNAs)

• Introduction to microRNA (miRNA)

• Role of miRNA in regulation of gene expression

• Non-coding RNA and RNA as genetic material in viruses

miRNA: Regulator of Gene Expression (Post-Transcriptional) - Detailed View

• miRNA genes are transcribed by RNA Polymerase II

• miRNA genes can reside within introns of protein-coding genes

• Mature miRNA are short (21–25 nt) single-stranded RNAs

• miRNA biogenesis involves processing by Dicer and loading into Argonaute (Ago) proteins to form RISC

• RISC (RNA-induced Silencing Complex) is the minimal complex for miRNA-mediated regulation; the miRNA acts as a guide to bring RISC to target mRNA via base pairing

• Target sites are commonly in the 3' UTR of the target mRNA; binding is not perfectly complementary; the seed sequence (nt 2–8) is the most critical region for initial binding

• Each miRNA is unique and can regulate multiple mRNA targets; one mRNA can have several miRNA binding sites

• miRNA regulation can repress translation (initiation or elongation) and/or promote mRNA degradation

• miRNA regulation serves as a fine-tuner of gene expression and is involved in a broad set of cellular processes

• miRNAs play roles in development, physiology, and disease; global decreases in miRNA expression have been observed in various cancers; some miRNAs act as tumor suppressors; others are upregulated and termed Oncomirs

• miRNA regulation provides rapid and broad regulatory potential due to the small size and multiple targets

miRNA Biogenesis and Functionally Relevant Complexes

• Mature miRNA bound to Argonaute forms the core of RISC

• RISC targets the mRNA via complementary sequences; the mechanism can include translational repression and/or mRNA degradation depending on availability and context

Non-Coding RNAs Beyond miRNA

• Long non-coding RNAs (lncRNA): >200 nt; transcribed by Pol II; similar processing to mRNA; diverse regulatory roles

• LncRNAs can act as miRNA sponges/decoys (ceRNA network), modulating miRNA availability for mRNA targets

• lncRNAs can regulate transcription by recruiting transcription factors or influencing chromatin structure (epigenetic regulation)

• Example: PTEN and its pseudogene lncRNA PTENP1 function as miRNA sponges; PTENP1 competes with PTEN mRNA for miRNA binding; when PTENP1 binds miRNA, PTEN translation is derepressed, increasing PTEN protein levels

ncRNA: Other Roles and Implications

• Non-coding RNAs are diverse in size, biogenesis, conservation, and function

• ncRNA research may illuminate human evolution, disease mechanisms, and genomic complexity

RNA as Genetic Material in Viruses (Revisited)

• RNA-based genomes are used by some viruses; this is a distinct role for RNA beyond cellular functions

• Examples and concepts include Rhinoviruses, influenza viruses, and Ebola virus as RNA viruses with RNA as their genetic material

Transcriptome and RNA Diversity in Humans

• The transcriptome is the complete collection of RNAs transcribed in a cell

• Only one RNA type codes for protein, i.e., mRNA; most RNAs are non-coding

• In humans, approximately 21,000 protein-coding genes are estimated; ~90% of the genome is transcribed, but only ~3% of transcribed RNA codes for proteins

Endnotes and Contextual Observations

• The regulatory capacity of RNA is vast, spanning transcriptional and post-transcriptional control

• RNA diversity supports complex regulation and cellular adaptability despite relatively compact genome sizes

• Splicing, alternative splicing, and ncRNA-mediated regulation collectively contribute to proteome diversity and organismal complexity