Transcription, RNA Processing, Non-Coding RNAs, and CRISPR-Cas: Mechanisms, Regulation, and Applications 9/4

Transcription: Overview and Key Components

• Goal: Convert a DNA template into an RNA transcript
• Core enzyme: RNA polymerase II (in eukaryotes) synthesizes RNA from a DNA template
• Direction of transcription: RNA synthesis occurs in the 5' → 3' direction; nucleotides are added to the 3'-OH of the growing RNA strand
  • $ext{RNA synthesis is in the } 5' <br /> ightarrow 3' ext{ direction, with new nucleotides added to the } 3'- ext{OH} ext{ of the growing chain}.$
• RNA produced is complementary to the DNA template strand and identical to the coding strand except for uracil replacing thymine
  • $ext{RNA sequence} = ext{coding strand sequence with } T <br /> ightarrow U.$
• Major RNA types and processing focus: messenger RNAs (mRNAs) and non-coding RNAs (miRNAs, siRNAs, etc.), with regulatory roles in transcription and post-transcriptional control
• Transcription initiation requires promoters and transcription factors; elongation and processing follow initiation

Promoters, General Transcription Factors, and Regulation of Transcription

• Promoter region is where transcription starts; it contains signals for RNA polymerase II binding
• TATA box: an AT-rich motif upstream of the start site that facilitates DNA melting
  • Binding protein: TATA-binding protein (TBP), part of the general transcription factor TFIID
• General transcription factors (GTFs): required at promoters universally
  • Key ones: TFII D (TFII-D, with TBP as a subunit) and TFII H
  • TFIIH role includes opening DNA and phosphorylating RNA Pol II, enabling promoter clearance
• RNA polymerase II requires assembly with GTFs and a mediator complex to form a transcription pre-initiation complex
  • Mediator: a stabilizing cofactor that helps enhancers communicate with the promoter; does not bind DNA itself but facilitates interactions
• Enhancers: distal regulatory elements that can be thousands of base pairs away from a promoter
  • Enhancers bind cell-type–specific transcription factors; looping of DNA brings enhancers in contact with the promoter–GTF–Pol II complex via mediator and other co-factors
• Specificity: General transcription factors are present in all cells; enhancer-bound transcription factors provide cell-type specificity
• Promoter architecture: promoter proximal elements, TATA box, and potential upstream regulatory sequences
• Transcription factors recognize specific DNA sequences without breaking base-pair hydrogen bonds; interactions are sequence-specific and often involve hydrogen bonding with bases in the major groove
• Some well-known transcription factors: SP1, ONC1, GATA1, MYOD, P53 (tumor suppressor; activates apoptosis in response to DNA damage)

RNA Polymerase II, Promoter Clearance, and CTD Phosphorylation

• Initiation: General transcription factors recruit RNA Pol II to the promoter
• TFIIH has two critical roles:
  • Pries apart DNA to enable transcription initiation and promoter melting
  • Phosphorylates the C-terminal domain (CTD) of RNA Pol II
• CTD phosphorylation facilitates promoter clearance and recruitment of RNA processing factors (capping, splicing, polyadenylation) as transcription proceeds
• Consequence of TFIIH dysfunction (hypothetical): without CTD phosphorylation, transcription initiation may occur at a low rate, but processing factors are not efficiently recruited, and RNA processing may be impaired
  • Result: transcripts may be retained in the nucleus due to failure to cap, splice, and polyadenylate properly and export

RNA Processing During Transcription: Capping, Splicing, and Polyadenylation

• Co-transcriptional processing: capping, splicing, and 3' end processing occur as RNA is synthesized
• 5' cap: early capping with a 7-methylguanosine cap
  • $5' ext{ cap} = ext{m}^7 ext{G}- ext{cap}$
  • Purpose: stabilize the transcript and facilitate ribosome recruitment for translation
• Splicing: removal of introns and joining of exons to produce mature mRNA
  • Introns are non-coding intervening sequences; exons encode the protein
  • Splicing is carried out by small nuclear ribonucleoproteins (snRNPs) and associated proteins (SNRNPs)
  • Mechanism: snRNPs recognize intron-exon boundaries, form base-paired interactions, and create a lariat intermediate; introns are removed and exons ligated
  • Exon Junction Complex (EJC): deposited after successful splicing; marks proper splicing and aids downstream export and translation
• 3' end processing: polyadenylation adds a poly(A) tail
  • Poly(A) tail length typical: ≈ $150-200 ext{ adenines}$
  • Poly(A) tail protects mRNA from degradation and aids export and translation
• Nuclear processing factors are recruited by CTD phosphorylation; capping factors engage early, splicing factors are engaged during elongation, and polyadenylation factors act at termination
• Nuclear export readiness: capped, properly spliced mRNA with EJC and poly(A) tail can be exported via the nuclear pore complex; binding proteins (cap-binding, poly(A)-binding proteins) and EJC influence export and translation readiness
• In bacteria (prokaryotes), genes typically lack introns; eukaryotic genes frequently contain introns and require RNA processing before export

Gene Regulation by Chromatin State and Enhancers

• Chromatin state controls accessibility of genes:
  • Euchromatin: loosely packed, transcriptionally active
  • Heterochromatin: tightly packed, transcriptionally repressed
• Only genes in euchromatin of a given cell type are generally transcribed
• DNA packaging and regulatory regions influence transcription likelihood; accessible chromatin allows promoter and enhancer recognition by transcription factors

Non-Coding RNAs and Post- Transcriptional Regulation

• Beyond mRNA, non-coding RNAs regulate gene expression at multiple levels
• MicroRNAs (miRNAs): small non-coding RNAs that regulate mRNA stability and translation
  • Biogenesis: transcribed in the nucleus, folded into hairpin structures, processed by Dicer, exported to cytoplasm, and loaded onto RISC (RNA-induced Silencing Complex)
  • Mature miRNA is typically single-stranded and guides RISC to target RNAs via base pairing
  • RISC contains Argonaute and other associated proteins; miRNA directs silencing by complementary binding to target mRNAs
  • Outcomes depend on complementarity:
  • Near-perfect match (often ~20 bases): endonucleolytic cleavage and rapid degradation of the target mRNA
    • Commonly described as mRNA cleavage by RISC-associated nucleases
  • Partial match (less extensive complementarity): translational repression and/or deadenylation leading to eventual degradation; mRNA may be sequestered in P bodies
  • Typical miRNA length: around 20–22 nucleotides
• Small interfering RNAs (siRNAs):
  • Often arise from double-stranded RNA and act as a primitive immune response against viral RNAs and transposons
  • Biogenesis: Dicer processing of dsRNA into siRNA duplexes; one strand is loaded into RISC
  • Cytoplasmic role: guide RISC to complementary RNAs for degradation
  • Nuclear role (via RITS): siRNA can guide transcriptional silencing in the nucleus by promoting heterochromatin formation and repressing transcription of transposons
• Long-term significance: non-coding RNAs act as adapters that recruit specific proteins to DNA or RNA, modulating transcription, RNA processing, stability, and translation
• Recap: miRNAs and siRNAs share: processing by Dicer, loading onto RISCs, cytoplasmic targeting of RNAs; but siRNA can also mediate nuclear transcriptional silencing via RITS

MicroRNAs and Small Interfering RNAs: Mechanisms and Pathways

• miRNA pathway:
  • Transcribed in the nucleus, formed into a hairpin precursor, processed by Dicer into mature miRNA, loaded into RISC
  • Targets mRNAs in cytoplasm; outcomes depend on base-pairing strength
• siRNA pathway:
  • dsRNA sensed by Dicer, processed to siRNA, loaded into RISC
  • Cytoplasmic degradation of complementary RNAs; nuclear silencing via RITS for retrotransposons
• P bodies: cytoplasmic mRNA regulatory sites where decapping and degradation enzymes localize; miRNA-mediated repression can recruit target mRNAs to P bodies for storage or decay
• Dual role of non-coding RNAs: act as adapters, bringing regulatory proteins to DNA/RNA and guiding modifications, degradation, or transcriptional changes

CRISPR-Cas: Gene Editing and Beyond

• CRISPR-Cas is a bacterial immune system adapted for genome editing
• Core concept: an RNA guide directs a Cas nuclease to a specific DNA sequence, creating a double-stranded break (DSB)
• Key components:
  • CRISPR locus: clustered repeats and spacers derived from past viral exposures; the spacer sequences guide targeting
  • Cas proteins: nuclease enzymes (e.g., Cas9) that cleave DNA at the target site guided by RNA
  • Guide RNA (gRNA): synthetic or natural RNA that is complementary to the target DNA and binds Cas9 to direct cutting
• Mechanism of editing:
  • Guide RNA binds Cas9 to form a complex that recognizes a complementary DNA sequence and induces a DSB
  • DNA repair pathways repair the break:
  • Homologous recombination (HR) with a donor DNA template allows precise sequence replacement or insertion
  • End-joining pathways (NHEJ) can introduce indels and disrupt the target gene
• Off-target effects: imperfect guide-DNA complementarity can cause unintended cuts elsewhere in the genome
• Applications and variants:
  • Gene knockout and mutation repair in cell lines and organisms
  • CRISPRa: catalytically dead Cas9 (dCas9) fused to transcriptional activators to upregulate gene expression
  • CRISPRi: dCas9 fused to repressors to downregulate gene expression
  • Therapeutic potential: trials in hematopoietic and some other contexts; ethical and regulatory considerations are paramount
• Historical context and caveats:
  • CRISPR originated from bacterial defense against phages; Jennifer Doudna and Emmanuelle Charpentier shared Nobel Prize in 2020 for CRISPR-Cas9 genome editing
• Ethical considerations: responsible use, governance, and potential societal impacts (e.g., germline editing, access disparities, unintended consequences)

Connections to Other Topics and Real-World Relevance

• Connections to sequencing and PCR: foundational techniques that enable gene editing, RNA analysis, and understanding gene expression
• Cancer biology: transcription factors like P53 regulate apoptosis and cell cycle; mutations have profound implications for cancer development and therapy
• Gene regulation principles: chromatin state, enhancer looping, and transcriptional regulation underpin development, differentiation, and disease
• Nobel Prize context: major breakthroughs in transcription regulation, sequencing, and CRISPR have reshaped biology and medicine
• Practical implications: RNA processing steps affect gene expression levels and quality of transcripts; CRISPR applications span research, therapy, and biotechnology

Quick Summary and Study Prompts

• Transcription basics: Promoter, TATA box, TBP, TFIIH; Pol II initiation, promoter clearance, CTD phosphorylation
• RNA processing: capping, splicing (introns/exons, SNReNPs, EJC), polyadenylation; nucleus export dependent on processing
• Non-coding RNAs: miRNAs and siRNAs regulate mRNA stability and transcription via RISC and RITS; roles in immune defense and genome stability
• CRISPR-Cas: guide RNA directs Cas9 to DNA; DSB repair via HR with donor DNA; off-target concerns; CRISPRa/CRISPRi as regulatory tools; ethical considerations
• Conceptual links: chromatin state controls gene accessibility; enhancer-promoter looping increases transcription; transcription factors provide tissue-specific patterns

Exam-Style Questions to Test Understanding

• Explain why transcription in eukaryotes requires both general transcription factors and RNA polymerase II, and describe the role of TFIIH in promoter clearance.
• Describe the sequence of co-transcriptional RNA processing events and why each step is important for mRNA export and translation.
• Compare and contrast miRNA and siRNA pathways, including their biogenesis, mechanism of action, and subcellular targets.
• Outline how CRISPR-Cas9 edits a gene and the differences between using HR versus NHEJ for repairing a DSB. Include potential off-target effects and how researchers attempt to minimize them.
• Discuss how enhancer elements regulate transcription from distant sites and how DNA looping facilitates this process.
• What would be the cellular consequence if TFIIH were nonfunctional and could not phosphorylate the RNA Pol II CTD? Explain in terms of transcription initiation, RNA processing, and nuclear export.

Key Terms to Memorize

• RNA polymerase II, TFIID, TFIIH, CTD phosphorylation, promoter, TATA box, enhancer, mediator, euchromatin, heterochromatin
• Cap: $ext{m}^7 ext{G-cap}$, splicing, intron, exon, exon junction complex (EJC), poly(A) tail
• miRNA, siRNA, RISC, Dicer, Argonaute, P bodies, RITS
• CRISPR, Cas9, guide RNA, double-strand break, homologous recombination, off-target effects
• CRISPRa, CRISPRi, dCas9, transcriptional regulation