Seminario 5 Regulación de la expresión génica II - Sebastian Giusti

Analysis of Gene Expression Regulation: Post-Transcriptional, Translational, and Post-Translational Levels

Overview of Today's Class

• The class focuses on the second part of the conceptual unit regarding gene expression regulation, specifically post-transcriptional, translational, and post-translational levels.

• A recap of previous discussions highlights initial steps in gene expression regulation, particularly chromatin condensation's role in transcription machinery accessibility.

Key Mechanisms in Gene Expression Regulation

• Molecular mechanisms regulating transcription initiation were discussed, emphasizing specific transcription factors that bind to DNA with co-regulators to recruit epigenetic modifiers.

• These modifiers include histone-modifying complexes that can acetylate or methylate histones and chromatin remodeling complexes that expose specific DNA segments based on enhancer or silencer interactions.

Transition from Transcription Initiation to Other Regulatory Levels

• The need for additional molecular signals for RNA polymerase II activation was emphasized; it requires a relaxed chromatin state at the promoter site.

• The previous class focused on how these processes determine whether a gene is expressed and its transcription frequency affecting product quantity.

Focus Areas for Today’s Seminar

• Today's seminar will delve into regulatory mechanisms following transcription initiation, including RNA processing and export to the cytosol.

• Stability of RNA molecules will be analyzed alongside protein stability and activity regulation; emphasis will be placed on messenger RNAs (mRNAs).

Properties of RNA Molecules

• RNA molecules are highlighted as labile compared to stable DNA; they degrade easily under various conditions both inside and outside cells.

• Inside cells, RNA is subject to degradation by ribonucleases which can act as exonucleases or endonucleases breaking down nucleotides.

Structural Characteristics of RNA

• Unlike linear representations in textbooks, intracellular RNAs adopt complex three-dimensional conformations through intramolecular base pairing.

• This structural complexity allows for specific recognition between RNA molecules and proteins based on their shapes.

Complementarity Between Proteins and RNAs

• The importance of three-dimensional complementarity between proteins binding to specific RNAs was noted; this mirrors enzyme-substrate interactions.

Processing of Eukaryotic mRNA

Overview of RNA Processing

• The three-dimensional structure of RNA allows it to bind with proteins and other nucleic acids through base complementarity, facilitating molecular recognition.

• Eukaryotic RNA processing, or maturation, involves modifications to the primary transcript produced by RNA polymerase II, transforming it into mature mRNA ready for cytosolic export.

Steps in mRNA Processing

• The processing includes three key steps:

• Modification of the 5' end (capping).

• Splicing of exons.

• Covalent modification of the 3' end.

• Research from the 1980s and 1990s revealed that these processing steps are co-transcriptional, occurring simultaneously with transcription by RNA polymerase II.

Enzymatic Associations

• Enzymes responsible for capping, splicing, and modifying the 3' end are associated with regions of RNA polymerase II during transcription.

Capping Process

• The first step is capping (5' modification), where a modified nucleotide is covalently attached to the first nucleotide introduced by RNA polymerase.

• This cap differs from regular nucleotides as it connects in a unique manner (5' to 5') and features a methylated guanine.

Functions of the Cap Structure

• The cap structure is recognized by cap-binding complexes (CBC), which protect the mRNA from degradation and facilitate its export through nuclear pores.

Modification of the 3' End

Polyadenylation Signal

• Within transcribed sequences, there exists a polyadenylation signal that triggers cleavage by enzymes traveling with phosphorylated CTD domain of RNA polymerase II.

• After cleavage, an enzyme called poly(A) polymerase adds adenine nucleotides to form a poly(A) tail at the transcript's end.

Importance of Poly(A) Tail

• This tail is not encoded in DNA but added enzymatically; it protects against exonuclease activity and aids in proper export and translation machinery recognition.

Splicing Mechanism

Understanding Gene Structure

• Eukaryotic genes are discontinuous; this discovery challenged previous understandings based on prokaryotic systems where genes are continuous.

Understanding Eukaryotic Gene Structure and Splicing

Discontinuous Nature of Eukaryotic Genes

• Eukaryotic genes are characterized by discontinuity, meaning that the sequences present in the mature transcript are not continuous in the genome. Instead, they are fragmented and interspersed with non-coding DNA.

Exons and Introns

• The coding sequences that remain in the final transcript after processing are called exons, while the intervening sequences that are removed during this process are known as introns. Notably, exons tend to be smaller than introns.

Molecular Process of Intron Removal

• The removal of introns is a highly precise molecular process that was better understood through research advancements from the 1970s onward. It involves cleaving covalent bonds at specific points in the primary transcript and joining two separated exons.

Importance of Accuracy in Splicing

• Any errors during splicing, such as a single nucleotide mistake, can lead to significant cellular consequences by altering codon reading frames during translation.

Mechanisms Guiding Splicing

• The scientific community sought to understand how intron removal occurs with such precision. Key to this process are consensus sequences within the primary transcript that guide molecular machinery responsible for splicing.

Key Sites for Splicing Recognition

• There are three critical regions involved in splicing:

• The donor site (5' end of an exon)

• The acceptor site (3' end of an intron)

• A branch point nucleotide within an exon

Role of Spliceosome Complex

• Discovered in the 1980s, spliceosomes consist of proteins associated with small nuclear RNAs (snRNAs), forming ribonucleoproteins essential for executing splicing steps accurately.

Composition and Functionality of Ribonucleoproteins

• Five distinct ribonucleoproteins make up the spliceosome, each linked to a small nuclear RNA (snRNA). These complexes facilitate recognition between exonic and intronic sequences through base complementarity.

Detailed Mechanism of Splicing Events

• Each splicing event removes one intron via two sequential reactions known as transesterifications:

• Initial cleavage occurs at the donor site where a branch point nucleotide forms a bond with an adjacent nucleotide.

• A second reaction links remaining exonic ends together after removing the intron loop.

Catalytic Role of Small Nuclear RNAs

Splicing Mechanisms and Protein Isoforms

Understanding Splicing and Its Implications

• The splicing process connects two hexons that were previously separated by an intron, leaving a protein residue at the site of splicing, represented in the diagram as a small red square known as the exon-binding complex.

• It is important to remember this molecular component as it will play a role in later analysis. While splicing occurs during transcription of any eukaryotic RNA, it does not happen uniformly across different tissues or developmental stages.

• Certain sequences within genes can behave differently depending on tissue type; for instance, exon three may be present in mature mRNA in one tissue but act as an intron in another.

• This variability leads to different tissues producing isoforms of the same protein, which are functional variants derived from the same gene. An example is tropomyosin, which plays a role in muscle contraction.

• The expression of tropomyosin varies across tissues; comparing exons between striated muscle and brain transcripts reveals differences in how these sequences function.

Alternative Splicing: Mechanisms and Regulation

• Alternative splicing increases genomic coding capacity by allowing different primary transcripts to be generated from the same DNA segment based on tissue type or developmental stage.

• This concept challenges traditional definitions of exons and introns since some sequences may act as exons in certain contexts but not others.

• Not all alterations to splicing mechanisms have been empirically observed; commonly noted is "exon skipping," where an exon behaves like an intron under specific conditions.

• Other mechanisms include intron retention, where typically removed introns remain present in some tissues. Understanding these requires examining molecular mechanisms behind alternative splicing.

• Consensus sequences at splice sites show high similarity but are not identical across all human genes. Some sequences have greater affinity for recruiting spliceosomal machinery than others.

Role of Regulatory Proteins

• Strong splice sites effectively recruit spliceosomes while weak sites require additional proteins for effective recruitment.

• In different tissues expressing the same gene (e.g., tropomyosin), strong splice sites can lead to successful intron removal if no blocking proteins are present.

• Conversely, if a regulatory protein binds to consensus sites at weak splice locations, it can prevent proper recognition by the spliceosome, resulting in retained introns behaving like exons instead.

Splicing and mRNA Processing

The Role of Splicing in Gene Expression

• In the absence of specific proteins, introns may not be removed during splicing, leading them to behave like exons and remain in the mature transcript.

• Alternative splicing occurs due to varying regulatory protein repertoires across different tissues or developmental stages, affecting gene expression.

mRNA Structure Post-Processing

• After processing steps (5' capping, 3' modification, and splicing), mature mRNA contains a 5' cap followed by untranslated regions (UTRs).

• The 5' UTR precedes the start codon (AUG), which codes for methionine; it is crucial for translation initiation.

• Following the stop codon in mRNA, there exists a 3' UTR that plays a role in transcript stability.

Importance of UTR Regions

• Both 5' and 3' UTR regions are essential for regulating mRNA stability; however, this idealized representation does not reflect cellular realities.

Protein Interactions with mRNA

• Mature RNA associates with various proteins acquired during maturation, including CAP-binding complexes and poly-A binding proteins.

• Nuclear proteins facilitate proper export of mature mRNA from the nucleus to the cytosol through nuclear pores.

Translation Initiation Factors

• Eukaryotic translation initiation factors interact with both CAP-binding complexes and poly-A binding proteins to initiate translation effectively.

• This interaction creates a circular configuration of mRNA that enhances translational efficiency.

Quality Control Mechanisms in Gene Expression

• The circular configuration acts as a quality control mechanism preventing truncated transcripts from being translated if they are damaged.

Ribosome Assembly During Translation

• Once in the cytosol, circularized mRNA allows ribosomal subunits to assemble efficiently around the start codon for translation.

Degradation of Faulty Transcripts

• If premature stop codons arise due to mutations, these faulty RNAs undergo degradation rather than producing truncated proteins.

Nonsense Mediated Decay Mechanism

• Understanding nonsense-mediated decay involves comparing normal transcripts with those containing premature stop codons during initial ribosomal translation.

Understanding mRNA Stability and Regulation

The Role of Exon Junction Complexes

• Exon junction complexes (EJC) stabilize transcripts by remaining attached at splice junctions between adjacent exons, ensuring transcript integrity.

• Early stop codons can cause ribosomes to detach prematurely, preventing modifications to downstream EJC, leading to early degradation of the transcript.

Mechanisms of mRNA Degradation

• Not all mRNAs have the same lifespan; some degrade quickly while others are more stable, indicating differential regulation within cells.

• Unstable mRNAs may last only a few minutes, while stable ones can persist longer. This variability is crucial for cellular function.

Factors Influencing mRNA Lifespan

• Proteins that control cell cycle often correspond with unstable mRNAs, whereas structural proteins tend to have longer half-lives.

• RNA binding proteins (RBPs) play a significant role in determining the stability of specific transcripts based on their three-dimensional structure.

MicroRNAs and Post-transcriptional Regulation

• MicroRNAs (miRNAs), small non-coding RNAs about 21 nucleotides long, regulate gene expression post-transcriptionally by guiding silencing complexes to target mRNAs.

• miRNAs bind to complementary sequences in the untranslated regions (UTRs) of target mRNAs, influencing their stability and translation efficiency.

Implications of miRNA Functionality

• The interaction between miRNAs and their targets leads to repression of translation and degradation of the messenger RNA.

• Human genome contains approximately 2,300 genes coding for different miRNAs which can regulate multiple mature transcripts with complementary sequences.

Evolutionary Significance of RNA Interference

• Unlike transcriptional controls that act as binary switches for gene expression, miRNAs fine-tune expression levels akin to equalizers.

• The discovery of RNA interference in the 1990s led to advancements in designing small artificial RNAs for therapeutic purposes.

Protein Activity Regulation Post-Synthesis

• After protein synthesis concludes in the cytosol, many proteins require specific conditions or modifications (e.g., phosphorylation or ligand binding) for activation.

Cell Cycle Regulation and Post-Translational Modifications

Mechanisms of Cell Cycle Regulation

• The activation of helicases at replication origins occurs only when they are phosphorylated by cyclin-dependent kinase (CDK) complexes, which are characteristic of the F phase.

• Post-translational modifications, such as phosphorylation, have immediate effects on cellular functions, typically within seconds to minutes, contrasting with slower modifications that may take hours.

Ubiquitin-Proteasome System

• Protein half-life is actively regulated by cells through the ubiquitin-proteasome system; ubiquitin is a small protein that can be conjugated to various substrates.

• The process of polyubiquitination involves multiple enzymes: E1 (activating enzyme), E2 (conjugating enzyme), and E3 (ligase), which specifically recognizes substrates for ubiquitination.

Specificity in Ubiquitination

• Different classes of E3 ligases exist across cell types, providing specificity in recognizing distinct substrates for polyubiquitination.

• E3 ligases can be inactive or active depending on cellular conditions like phosphorylation, influencing whether certain substrates undergo polyubiquitination.

Consequences of Polyubiquitination

• The addition of a polyubiquitin chain leads to protein degradation by proteasomes, which recognize extensively polyubiquitinated proteins as substrates.

• Cyclins are frequently degraded via this mechanism; their levels fluctuate throughout the cell cycle due to signals that activate specific E3 ligases.

Summary of Gene Expression Regulation

• Gene expression regulation encompasses mechanisms controlling both the quantity and activity level of gene products. Pre-transcriptional regulation affects product presence while post-translational modifications influence activity.