Workshop Week 31_lncRNAs

Page 7: Basic Principles of Gene Regulation

• Gene expression regulated by various factors that interact with DNA or mRNA.

• Regulators can repress or activate targets.

• Regulators may be proteins or RNA.

• RNA can control gene expression through secondary structures (inter- or intramolecular).

Page 8: The Size of the Non-Coding Genome

• Human Genome Project (1996-2001) aimed to sequence the human genome and identify human genes.

• Quote by President Bill Clinton: "Today we are learning the language in which God created life".

Page 9: Human Genome Composition

• Only 1% of the human genome consists of exons.

• Considerations of the function of 'junk DNA' or 'dark matter of the genome'.

Page 10: ENCODE and ModENCODE

• ENCODE: Encyclopedia of DNA Elements (H.sapiens) aims to delineate functional elements of the genome.

• ENCODE project focuses on transcription factors, chromatin modifications, DNA methylation, enhancer discovery, transcriptomics.

• Discovery of many non-coding RNA species highlighted.

Page 11: RNA Profiling Insights

• Genome-wide profiling shows most of the genome is transcribed including gene regions and non-coding regions.

• 70% of human genes produce antisense RNA.

Page 12: DNA-RNA-Proteins Comparison

• Non-coding vs coding RNAs.

• Presence of approximately 20,000 proteins in H.sapiens.

Page 13: Non-Coding RNA Classification

• Non-coding RNAs categorized based on length (long > 200 bp, short 20-30 bp).

• Focus on long non-coding RNAs (lncRNAs) in this lecture.

Page 14: Long Non-Coding RNAs in the Genome

• Over 60,000 lncRNAs identified through sequencing and bioinformatic analysis.

• They show little sequence conservation across species.

• Locations in the genome include intergenic regions, telomeres, enhancers, introns, and exons.

Page 15: lncRNA Sequence and Structure

• LncRNAs are produced by RNA-Polymerase II, can undergo splicing and polyadenylation (not always).

• Formation of complex secondary structures is common.

• Often derived from antisense strands.

Page 16: Antisense RNA Production

• Many lncRNAs are antisense RNA transcribed from sense gene bodies, with ~70% of mouse genes producing antisense transcripts.

• Antisense transcription can inhibit mRNA translation and degrade mRNA to knock down gene function.

• Potential to enhance translation as well as interact with DNA binding factors.

Page 17: Mechanisms of lncRNA Action

• lncRNAs control gene expression transcriptionally (DNA-Chromatin) and post-transcriptionally (RNA-Cytoplasm).

• Interactions can occur with RNA, DNA, and proteins.

Page 18: Examples of lncRNAs and Their Functions

• Request for real examples of lncRNAs discussed in the workshop.

Page 19: HOTAIR lncRNA

• HOTAIR recruits a repressive chromatin remodeling complex to the HoxD-cluster (antisense lncRNA).

• Functions in trans by recruiting PRC2, leading to reduction in HoxD transcription upon knockdown.

Page 20: HOTAIR in Tumor Growth

• Overexpression of HOTAIR correlates with tumor growth and metastasis in breast cancer.

• High levels of HOTAIR in patients predict worse prognosis.

Page 21: Role of Braveheart lncRNA

• Braveheart involved in cardiomyocyte differentiation by binding and titrating the TF CNBP, which inhibits heart formation.

Page 22: Braveheart and Chromatin

• Braveheart facilitates an open chromatin state by removing PRC2 from regions of the genome, allowing for chromatin activation.

Page 23: Xist in X-Chromosome Silencing

• Overview of X-inactivation in females, involving lncRNA Xist.

• Xist contributes to the condensation of the inactive X-chromosome into a repressive state.

Page 24: Xist Recruitment Mechanism

• Xist is exclusively localized to the inactivated X-chromosome and recruits HDACs and PRC2.

• Alters chromatin structure by aggregating loops at the nuclear envelope.

Page 25: Summary of lncRNA Functions

• lncRNAs regulate gene transcription and translation through multiple mechanisms involving chromatin remodeling, transcription factors, and mRNA interactions.

Page 26: Take-home Points on lncRNAs

• lncRNAs are abundant (>60,000) and often antisense transcribed.

• They play key roles in regulating transcription, translation, and chromatin structure in development and cancer.

Page 27: Upcoming Practical

• Next week: Practical RNAi session with C. elegans.

• Scheduled for Monday and Thursday with Dr. Jennifer Tullet.

• Further information to be provided on Moodle.

Page 28: Suggested Reading

• Essential Genes, 4th edition: Chapter 27 (Noncoding RNA).

• Genes XII: Chapter 29 (Noncoding RNA).

• Additional resource: iBiology lecture by Howard Chang on Xist lncRNA (available on Youtube).