MicroRNAs

Unit 10: MicroRNAs in Cellular Stress Responses

Overview of MicroRNAs (miRNAs)

  • Definition of miRNAs: MicroRNAs (miRNAs) are small RNA molecules that play a crucial role in the regulation of gene expression.

    • They are non-coding RNAs, meaning they do not code for proteins.

    • Length of miRNAs: Approximately 18–26 nucleotides long.

    • Some miRNAs are found in the introns of protein-coding genes (~30% of known miRNAs).

  • Significance of miRNAs:

    • They control how many proteins are made by other protein-coding genes.

    • One miRNA can regulate hundreds of different mRNAs.

  • Historical Context:

    • Discoveries by Victor Ambros and Gary Ruvkun demonstrated the role of miRNAs as negative regulators in gene expression, specifically their work on lin-4 and lin-14 in C. elegans.

Biogenesis of MicroRNAs

  • Transcription Process:

    • miRNAs are transcribed from DNA by RNA Polymerase II (RNA Pol II), yielding primary miRNA transcripts (pri-miRNA).

    • Pri-miRNA folds into a stem-loop structure (hairpin structure).

  • Processing Steps:

    1. Initial Cleavage: Endonuclease enzyme Drosha cleaves pri-miRNA to produce a pre-miRNA (~70 nucleotides).

    2. Transport: Exportin5 exports pre-miRNA from the nucleus to the cytoplasm.

    3. Final Processing: In the cytoplasm, Dicer further processes pre-miRNA into a double-stranded miRNA (miRNA-miRNA* duplex), each strand being ~22 nucleotides.

  • Formation of miRNA Complexes: One of the mature strands binds to Argonaute, a core protein of the RNA-induced silencing complex (RISC).

    • RISC complexes associate with target mRNAs at the 3’ untranslated region (3’UTR).

Function of MicroRNAs in Cellular Stress Responses

  • Role of miRNAs under Stress:

    • miRNAs are involved in the regulation of gene expression patterns in response to various stressors such as oxidative stress, hypoxia, heat shock, and nutrient deprivation.

  • Mechanisms of Action:

    • Changes in miRNAs can modulate gene expression in a stress context.

    • Cells may experience a phenotypic crisis under stress when miRNAs are mutated, highlighting their critical role in stress responses.

Mechanisms of miRNA Target Recognition and Regulation

  • Target Gene Interaction: Over 60% of mammalian genes are predicted targets of miRNAs.

    • A single miRNA can interact with multiple target genes, influencing various cell functions.

  • Seed Sequence:

    • Critical for target selection; defined as the first 2–8 nucleotides at the 5’ end of the miRNA.

    • Types of Seed Matches:

    • 6mer: A perfect match of six nucleotides.

    • 7mer-m8: Perfect match from nucleotides 2–8, plus an A across from nucleotide 1.

    • 7mer-A1: Include additional matches as above but differ slightly in nucleotide inclusion.

    • 8mer: A complete match from nucleotides 2–8 with an additional A across from nucleotide 1.

  • Complementarity Outcomes:

    • Perfect complementarity leads to mRNA degradation.

    • Imperfect complementarity inhibits protein synthesis.

Changes in miRNA Expression Under Stress

  • Experimental Evidence: Example of hypoxia altering miRNA expression in HT29 cells (colon cancer cell line), shown on a heat map indicating a time-dependent expression change.

  • Reciprocal Relationships: Between miRNA levels and target mRNA expression:

    • Increase in specific miRNAs (e.g., miRNA C) leads to decreased mRNA levels.

    • Decrease in other miRNAs (e.g., miRNA B) can lead to increased target mRNA expression.

Target Expression Modulation Mechanisms

  • Target Mimicry:

    • miRNAs can interact with mimics of target mRNAs, leading to competition for binding sites.

    • The expression of mRNA targets can be relieved when mimic levels increase during stress.

  • Creation/Deletion of Binding Sites:

    • Stress can lead to differential isoform expression resulting in either the creation or deletion of miRNA binding sites, affecting target expression levels.

Activity of miRNA-Protein Complexes

  • Complex Formation: miRNAs associate with various RNA-binding proteins (e.g., Argonaute) influencing miRNA function under stress.

  • Posttranslational Modifications: Changes can arise from direct interactions with cofactors, affect the activity levels of the RISC complex, or lead to sequestration to distinct cellular locations.

Changing Modes of miRNA Action Under Stress

  • Balance Between Degradation and Inhibition: Stress can shift the action of miRNA-protein complexes towards either promoting mRNA degradation or inhibiting translation.

  • Intracellular Granules:

    • P-bodies: Sites for mRNA decay, containing Argonaute proteins and degradation machinery.

    • Stress Granules: Cytoplasmic aggregates stalled in the early translation process, significant for mRNA storage and stress signaling.

Insights into Eukaryotic Gene Expression Regulation

  • Mechanisms of Gene Expression Control: Transcription factors and miRNAs are two main regulatory components.

    • Transcription Factors (TF) can bind regulatory sequences and influence gene expression positively or negatively.

  • Interactions Between TFs and miRNAs:

    • The interplay can lead to various timing patterns for gene expression responses to stress (e.g., pulse, lag, decrease, reinforced decrease).

Timing Patterns of Target mRNA Expression

  • Stability of miRNAs: Mature miRNAs have a long half-life (e.g., 12 days in vivo), accumulating over time upon activation.

  • Immediate Response of mRNAs: Target mRNAs can exhibit immediate expression changes, creating complex time-dependent dynamics in gene expression in response to stress.