MicroRNA and Gene Regulation in C.elegans

C.elegans

• lin-4 gene encodes microRNAs
• lin-4 microRNA is complementary to the 3’UTR of lin-14 mRNA
• lin-4 microRNA inhibits gene expression by triggering degradation of lin-14 mRNA

Lin-14

• Transcription factor required during the first larval phase
• Lin-14 is not needed afterward
• Binding of small RNAs (microRNAs) to repetitive sequences in the 3’UTR of lin-14 mRNA triggers degradation of transcripts

MicroRNAs

• “Hairpin-loop” structures trigger a protective mechanism - RNA interference
• RNA interference inhibits transcription and translation of genes
• Target sequence recognition depends on the strength of miRNA complementarity

Drosha

• Drosha is the enzyme that makes individual pre-miRNA hairpins.

RISC Complex

• RISC: RNA-induced silencing complex
• RISC separates dsDNA strands and aligns with the 3’UTR of the target mRNA
• Cleaves the mRNA or blocks translation

Basic Tools of Developmental Genetics

In Situ Hybridization

• Used to determine when and where genes are expressed in an embryo
• Example: Localizing odd-skipped gene expression in Drosophila by in situ hybridization
• RNA probe against odd-skipped mRNA is designed and used for hybridization and immunodetection

Chromatin Immunoprecipitation-Sequencing (ChIP-Seq)

• Determines where transcription factors bind and the location of modified nucleosomes

CRISPR/Cas9

• Used to test gene function by introducing gene edits

GAL4-UAS System

• Used to test gene function by controlling gene expression in a specific tissue or cell type.

Cre-Lox System

• Allows for the deletion or recombination of specific DNA sequences in a tissue-specific manner

Specifying Cell Identity

Cell Fate Specification

• Autonomous: cell fate is determined by intrinsic factors within the cell itself
• Conditional: cell fate depends on interactions with neighboring cells or the environment
• Syncytial: cell fate is determined by gradients of signaling molecules within a common cytoplasm

Single-Cell RNA Sequencing

• Used to map cell fates by determining which genes are expressed at any given time
• Helps to define cell identity

GFP and lacZ Reporters

• GFP reporter fused to retina-specific gene in zebrafish
• lacZ reporter fused to neuron-specific L1 gene in mouse

Differentiation

• Cell stops dividing and develops specialized structural elements and functional properties

Autonomous Specficiation

• Conklin described autonomous specification of muscle cells in tunicate blastomeres
• Removal of B4.1 blastomere results in a larva with no tail muscles
• Whittaker found that Macho mRNA segregates asymmetrically in the cytoplasm to drive tail muscle development

Conditional Specification

• Driesch’s experiments: conditional specification in sea urchin embryos
• Blastomeres have greater potential for cell fates than expected during normal development
• Cell-cell interactions are critical for normal development

Syncytial Specification

• Syncytial blastoderm in Drosophila
• Morphogen gradients specify syncytial cell fates
• Opposing gradients of Bicoid and Caudal transcription factors specify segment identities
• Autonomous: Transcription factors are differentially expressed after cellularization
• Conditional: Position relative to neighboring nuclei determines cell fate

Epigenetic Modifications

• Modulate access to genes and influence transcription
• Nucleosomes and chromatin structure play a role in epigenetic regulation

Histone Modifications

• Histone methylation can prevent access to promoters and block transcription
• Histone acetylation exposes DNA to RNA polymerase II and transcription factors, activating transcription

DNA Methylation

• DNA methylation blocks transcription by preventing the binding of transcription factors
• Loss of MeCP2 (methyl CpG-binding protein 2) leads to Rett’s syndrome in females. Symptoms: loss of language, difficulty walking, repetitive hand movements.

Transcription Factors

• Transcription factors regulate gene transcription by binding to specific DNA sequences
• Transcription factors in a given family share DNA-binding domains
• Yamanaka factors can induce pluripotent stem cells from differentiated skin cells

Pre-mRNA Processing: Alternative Splicing

• Differential pre-mRNA processing can produce a wide variety of proteins from the same gene
• Splicing: cut, rearrange, and ligate exons together
• Protein isoforms can play similar roles in different cells or contrasting roles in the same cell
• Dscam gene in Drosophila contains 115 exons and can generate 38,016 splice isoforms
• Dscam encodes a membrane adhesion protein essential for self-avoidance between dendrites from the same neuron
• Mutations in the Dscam homologue can contribute to the neurological defects associated with Down syndrome.

mRNA Translation: Ribosomal Selectivity and Cytoplasmic Localization

• Mouse: selective activation of translation of specific mRNAs
• Drosophila egg: mRNA localization in the cytoplasm
• Nanos mRNA diffuses and is trapped by anchor proteins at the posterior pole
• Hsp83 mRNA is degraded everywhere except at the posterior pole, where it is protected by protein complexes
• Bicoid and Oscar mRNAs are actively transported by motor proteins to the posterior and anterior poles, respectively
• Rpl38 deficiency: cannot translate subset of Hox genes to specify vertebrae → deformed skeleton

MicroRNA Regulation of mRNA Transcription and Translation

• MicroRNAs (miRNAs) regulate gene expression by binding to target mRNAs
• MicroRNAs can either block translation or trigger mRNA degradation
• The strength of the complementarity between the miRNA and its target mRNA determines the regulatory outcome
• MicroRNAs play crucial roles in various biological processes, including development, differentiation, and disease.

This quiz explores the role of microRNAs, specifically the lin-4 gene, in gene regulation during the development of C.elegans. Learn how microRNAs interact with mRNA and their significance in RNA interference and developmental genetics.