🔬Biological Chemistry I Unit 13 – RNA Transcription and Processing

RNA Basics

• RNA (ribonucleic acid) consists of a single-stranded molecule composed of nucleotides
  • Each nucleotide contains a ribose sugar, a phosphate group, and one of four nitrogenous bases (adenine, guanine, cytosine, or uracil)
• RNA plays a crucial role in the central dogma of molecular biology, acting as a messenger between DNA and proteins
• RNA is synthesized from a DNA template through the process of transcription, which is carried out by RNA polymerases
• Unlike DNA, RNA contains the sugar ribose instead of deoxyribose and the base uracil (U) instead of thymine (T)
• RNA molecules are typically shorter than DNA molecules and are more prone to degradation due to the presence of the 2'-OH group on the ribose sugar
• RNA can form secondary structures, such as hairpins and loops, through base pairing interactions (A-U and G-C)
• The three main types of RNA are messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA), each with distinct functions in gene expression

Transcription Initiation

• Transcription initiation is the first step in the synthesis of RNA from a DNA template, which occurs in the nucleus of eukaryotic cells
• RNA polymerase recognizes and binds to specific DNA sequences called promoters, which are located upstream of the gene to be transcribed
  • Promoters contain consensus sequences, such as the TATA box and the initiator element, that facilitate the binding of transcription factors and RNA polymerase
• Transcription factors, such as TFIID and TFIIB, assemble at the promoter region to form the pre-initiation complex (PIC)
• The PIC helps to recruit and position RNA polymerase at the transcription start site (TSS), which is typically located a short distance downstream of the promoter
• RNA polymerase unwinds the double-stranded DNA template, exposing the template strand for RNA synthesis
• The first nucleotide to be incorporated into the growing RNA chain is usually a purine (adenine or guanine) and is determined by the sequence of the DNA template
• Once the first few nucleotides are synthesized, RNA polymerase clears the promoter region and enters the elongation phase of transcription

Elongation and Termination

• During elongation, RNA polymerase moves along the DNA template in the 3' to 5' direction, synthesizing the complementary RNA strand in the 5' to 3' direction
• RNA polymerase catalyzes the formation of a phosphodiester bond between the 3'-OH group of the growing RNA chain and the 5'-phosphate group of the incoming nucleotide triphosphate (NTP)
• The elongation process continues until RNA polymerase encounters a termination signal, which can be either a sequence-dependent or a factor-dependent signal
  • Sequence-dependent termination involves the formation of a stable hairpin structure followed by a series of uracil residues, causing RNA polymerase to stall and dissociate from the DNA template
  • Factor-dependent termination requires the action of termination factors, such as Rho in prokaryotes, which bind to the nascent RNA and cause RNA polymerase to dissociate
• Once transcription is terminated, the newly synthesized RNA molecule is released from the DNA template and undergoes further processing steps
• The elongation rate of RNA polymerase varies depending on the organism and the specific gene being transcribed, but it typically ranges from 20 to 80 nucleotides per second in eukaryotes

Post-Transcriptional Modifications

• After transcription, the newly synthesized RNA molecule undergoes various post-transcriptional modifications that alter its structure, stability, and function
• One of the most common modifications is the addition of a 5' cap structure, which consists of a modified guanosine nucleotide attached to the 5' end of the RNA via a 5'-5' triphosphate linkage
  • The 5' cap protects the RNA from degradation by 5' exonucleases and facilitates its translation by eukaryotic ribosomes
• Another important modification is the addition of a poly(A) tail to the 3' end of the RNA, which involves the enzymatic addition of a series of adenine nucleotides
  • The poly(A) tail enhances RNA stability, facilitates nuclear export, and promotes translation efficiency
• RNA can also undergo base modifications, such as the conversion of adenosine to inosine (A-to-I editing) or cytidine to uridine (C-to-U editing), which can alter the coding potential or the stability of the RNA
• Some RNA molecules, particularly tRNAs and rRNAs, undergo extensive post-transcriptional modifications, including methylation, pseudouridylation, and the addition of various chemical groups
• These modifications can influence the structure, function, and interactions of the RNA molecules with other cellular components
• Post-transcriptional modifications are carried out by a variety of enzymes, such as methyltransferases, pseudouridine synthases, and RNA editing enzymes, which recognize specific sequences or structural motifs in the RNA

RNA Processing and Splicing

• In eukaryotes, most protein-coding genes are initially transcribed as precursor mRNAs (pre-mRNAs) that contain both exons (coding sequences) and introns (non-coding sequences)
• To generate mature mRNAs, the introns must be removed, and the exons must be joined together through a process called RNA splicing
• Splicing is carried out by the spliceosome, a large ribonucleoprotein complex composed of five small nuclear RNAs (snRNAs) and numerous protein factors
  • The spliceosome assembles on the pre-mRNA and catalyzes two transesterification reactions that excise the intron and ligate the flanking exons
• Splicing requires the recognition of specific sequences at the intron-exon boundaries, known as the 5' splice site (GU) and the 3' splice site (AG), as well as a branch point sequence located within the intron
• Alternative splicing is a mechanism by which different combinations of exons can be included or excluded from the final mRNA product, allowing a single gene to encode multiple protein isoforms
  • Alternative splicing is regulated by cis-acting regulatory elements (exonic splicing enhancers and silencers) and trans-acting factors (splicing regulators) that influence spliceosome assembly and splice site selection
• RNA editing, such as A-to-I editing, can also occur in conjunction with splicing, further increasing the diversity of the transcriptome
• Defects in RNA splicing can lead to various genetic disorders, such as spinal muscular atrophy and retinitis pigmentosa, highlighting the importance of accurate and efficient splicing for normal cellular function

Types of RNA and Their Functions

• Messenger RNA (mRNA) serves as the template for protein synthesis, carrying the genetic information from DNA to the ribosomes
  • mRNAs contain a 5' cap, a coding sequence (CDS) flanked by untranslated regions (UTRs), and a 3' poly(A) tail
• Transfer RNA (tRNA) acts as an adapter molecule that links the genetic code in mRNA to the amino acid sequence of proteins
  • tRNAs have a cloverleaf secondary structure and a 3' CCA end that is aminoacylated with a specific amino acid
  • The anticodon loop of tRNA base-pairs with the corresponding codon in mRNA during translation
• Ribosomal RNA (rRNA) is a structural and catalytic component of ribosomes, the cellular machinery responsible for protein synthesis
  • Eukaryotic ribosomes contain four rRNA molecules (28S, 18S, 5.8S, and 5S) that, together with numerous ribosomal proteins, form the 60S and 40S ribosomal subunits
• Small nuclear RNAs (snRNAs) are involved in the splicing of pre-mRNAs, forming the core components of the spliceosome (U1, U2, U4, U5, and U6 snRNAs)
• Small nucleolar RNAs (snoRNAs) guide the post-transcriptional modification of rRNAs and other non-coding RNAs, such as methylation and pseudouridylation
• MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally by base-pairing with complementary sequences in target mRNAs, leading to translational repression or mRNA degradation
• Long non-coding RNAs (lncRNAs) are a diverse class of RNA molecules (>200 nucleotides) that participate in various cellular processes, such as transcriptional regulation, chromatin remodeling, and RNA processing

Regulation of RNA Transcription

• The regulation of RNA transcription is crucial for controlling gene expression and ensuring the proper development and function of cells and organisms
• Transcriptional regulation is mediated by the interplay between cis-acting regulatory elements (promoters and enhancers) and trans-acting factors (transcription factors and chromatin modifiers)
• Promoters contain specific DNA sequences, such as the TATA box and the initiator element, that recruit the basal transcription machinery and determine the transcription start site
• Enhancers are distal regulatory elements that can activate transcription from promoters through long-range interactions mediated by chromatin looping
  • Enhancers contain binding sites for tissue-specific transcription factors that modulate gene expression in a cell type-dependent manner
• Transcription factors are proteins that bind to specific DNA sequences and regulate the activity of RNA polymerase II
  • Activators promote transcription by recruiting coactivators and chromatin remodeling complexes, while repressors inhibit transcription by blocking the binding of activators or recruiting corepressors
• Chromatin structure plays a critical role in transcriptional regulation, with open chromatin (euchromatin) being associated with active transcription and closed chromatin (heterochromatin) being associated with gene silencing
  • Histone modifications, such as acetylation and methylation, and DNA methylation are epigenetic marks that influence chromatin accessibility and transcription factor binding
• Transcriptional regulation can also be modulated by external signals, such as hormones, growth factors, and environmental stresses, which activate signal transduction pathways that converge on transcription factors
• Non-coding RNAs, such as miRNAs and lncRNAs, can also regulate transcription by targeting transcription factors or modifying chromatin structure
• Dysregulation of transcriptional control is associated with various diseases, including cancer, developmental disorders, and metabolic syndromes, emphasizing the importance of precise gene regulation for maintaining cellular homeostasis

RNA in Disease and Therapeutics

• Alterations in RNA metabolism, such as splicing defects, RNA editing abnormalities, and changes in RNA stability, can contribute to the development and progression of various diseases
• Mutations in splicing regulatory elements or splicing factors can lead to aberrant splicing patterns, resulting in the production of non-functional or deleterious protein isoforms
  • Spinal muscular atrophy is caused by mutations in the SMN1 gene that impair the splicing of SMN2, leading to reduced levels of functional SMN protein
  • Retinitis pigmentosa can be caused by mutations in splicing factors, such as PRPF31 and PRPF8, leading to defects in the splicing of retinal genes
• Alterations in RNA editing, particularly A-to-I editing, have been implicated in various neurological disorders, such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease
  • Reduced A-to-I editing of the glutamate receptor subunit GluA2 has been observed in ALS patients, potentially contributing to neuronal excitotoxicity
• Changes in RNA stability, mediated by mutations in RNA-binding proteins or alterations in miRNA expression, can lead to the accumulation of toxic RNA species or the depletion of essential proteins
  • Myotonic dystrophy is caused by the expansion of CUG or CCUG repeats in non-coding regions of the DMPK or CNBP genes, respectively, leading to the sequestration of RNA-binding proteins and widespread RNA processing defects
• RNA-based therapeutics have emerged as a promising approach for treating genetic diseases and cancer by modulating RNA metabolism or gene expression
  • Antisense oligonucleotides (ASOs) are short, synthetic nucleic acids that can bind to complementary sequences in target RNAs, promoting their degradation or modulating their splicing
    • Nusinersen is an FDA-approved ASO for the treatment of spinal muscular atrophy, which promotes the inclusion of exon 7 in SMN2 mRNA, increasing the production of functional SMN protein
  • Small interfering RNAs (siRNAs) are double-stranded RNA molecules that can silence gene expression by triggering the degradation of complementary mRNAs through the RNA interference (RNAi) pathway
    • Patisiran is an FDA-approved siRNA-based drug for the treatment of hereditary transthyretin-mediated amyloidosis, which reduces the production of mutant transthyretin protein by targeting its mRNA
• RNA-based vaccines, such as mRNA vaccines, have gained attention as a rapid and flexible platform for developing vaccines against infectious diseases, as demonstrated by the COVID-19 mRNA vaccines developed by Pfizer-BioNTech and Moderna
• The growing understanding of RNA biology and the development of novel RNA-targeting technologies hold promise for the discovery of new therapeutic strategies for a wide range of diseases