Non-Coding RNA and Gene Regulation

• The majority of RNA is non-coding but plays a key role in gene regulation.
• miRNA's basic structure, mechanism of biogenesis, and method of gene expression inhibition.
• miRNA's importance in normal development, disease treatment, and diagnosis.
• IncRNAs basic structure and their different mechanisms of gene regulation.
• The importance of IncRNA in normal development and disease.

Non-Coding RNA

• A large proportion of the human genome(>70%) is transcribed to RNA
• A small percentage (~3%) of RNA is translated into proteins
• The majority (~97%) of RNA doesn't encode for a protein, that is non-coding RNA
• Non-coding RNA is not "junk" and has important roles, including gene regulation.
• Broadly divided into two groups based on length: short non-coding RNA (sncRNA) - up to 200 nucleotides, or long non-coding RNA (IncRNA) – over 200 nucleotides

Short non-coding RNA

• microRNA (miRNA): Gene regulation
• Small interfering (siRNA): Germ-line cells – Genome defense
• Piwi-protein-interacting RNA (piRNA): Germ-line cells – Genome defense
• Transfer RNA (tRNA): Translation
• Small Cajal body RNA (scaRNA): snRNA maturation
• Small nucleolar RNA (snoRNA): Ribosomal RNA maturation
• Small nuclear RNA (snRNA): RNA splicing

Long non-coding RNA

• Nuclear IncRNA: Chromatin modification/Transcription
• Cytoplasmic IncRNA: Translation/Protein localization and activity
• Non-coding RNAs change gene expression without altering the DNA sequence which is epigenetic regulation.

MicroRNA (miRNA)

• Small non-coding RNAs that contain 10-25 nucleotides
• Transcribed from DNA sequences into primary miRNAs (pri-miRNAs) by RNA Polymerase II (like mRNAs)
• Processed into mature miRNAs (like mRNAs)
• Partially bind to mRNA in a sequence-dependent manner and silence gene expression
• One miRNA targets many different genes
• DNA sequences encoding for miRNAs are usually found within introns (non-protein coding regions of genes) or intergenic regions (regions between genes)
• miRNA genes are under the control of promoters and enhancers (like protein-encoding genes)
• Regulation of miRNA expression is similar to mRNA regulation

Regulation of miRNA Expression is similar to mRNA regulation

• Epigenetic/chromatin remodelling happens via Histone/DNA modifications
• Transcription is controlled by transcription factors
• Post-transcriptional is Regulation by RNA processing and editing
• A single-stranded pri-miRNA molecule folds into a stem-loop (hairpin) secondary structure
• Hairpin structure is required for pri-miRNA processing:

pri-miRNA processing:

• Recognition by ribonuclease III enzymes
• Ribonuclease III (RNAse III) – endonucleases that cleave dsRNA at specific locations
• RNA maturation
• G-U mismatched pairs (wobble pairs) in the stem of pri-miRNA are required.
• Maintaining the hairpin conformation
• Recognition and cleavage by RNAse III
• Recognition of target mRNA

miRNA processing:

• Drosha and Dicer are RNAse III endonucleases
• Cut dsRNA at specific locations
• Produce a short miRNA duplex with a 3' overhang

miRNA maturation:

• miRNA duplex associates with RNA-induced silencing complex (RISC)
• RISC unwinds the miRNA duplex
• miRNA strand (mature miRNA) guides RISC to target mRNA
• The other strand (passenger) is removed.
• mRNA is recognized by miRNA by a seed region.
• The seed region is nucleotides 2-8 on the 5' end of miRNA, it is essential for target recognition.
• miRNA binds to the 3'-UTR of the target gene mRNA by complementary base pairing
• It recognizes specific sequences called miRNA response elements.
• Additional base pairing at the 3' end is also possible.

Gene Silencing

• High complementarity of miRNA with mRNA leads to mRNA degradation
• It only happens rarely in human cells
• Low complementarity of miRNA with mRNA leads to translation inhibition
• It is most Common in human cells
• RISC promotes mRNA degradation by removing mRNA 3' poly-A tails and 5' cap. mRNA degrades by exonucleases
• RISC inhibits translation by preventing ribosome assembly, initiation factor binding, and translation elongation

RISC complex

• Dicer loads the miRNA duplex onto an Argonaute protein
• Unwinds the duplex into two strands.
• Removes the passenger strand/Guide strands recognise mRNA
• Binds other proteins, including GW182.
• GW182 is a scaffolding protein that recruits deadenylases and decapping factors – mRNA degradation, and translation inhibitors – Translation inhibition
• miRNAs regulate many different genes
• Each miRNA can regulate thousands of different genes

• 2,500 miRNAs in the human genome regulate >60% of genes. Can affect Cell metabolism, division, differentiation, and death

Regulation

• Different tissues express specific miRNAs
• miRNA expression is regulated during development (different miRNA levels at different developmental stages)
• miRNAs are crucial in normal development
• Abnormal changes (increased or decreased) in the expression of specific miRNAs can lead to diseases like Cancer, diabetes, Parkinson's, and Alzheimer's
• Restoring normal miRNA expression can be a potential therapy

Therapies

• miRNA mimics = synthetic double stranded RNA oligonucleotides that mimic endogenous miRNAs – increase endogenous miRNAs
• Anti-miRs = synthetic single-stranded RNA oligonucleotides that bind miRNAs and inhibit their action
• miRNA-targeting drugs have entered clinical trials, but none have been approved yet
• Dysregulated miRNAs act as causes of disease:
• Increase/decrease in tumor suppressor microRNAs
• dysregulation leads to tumorigenesis
• Circulating miRNA acts as disease biomarkers:
• miRNAs excreted into extracellular fluids
• Peripheral blood miRNAs are potential biomarkers for several diseases
• Can provide Accurate and less invasive diagnosis

Long non-coding RNA (IncRNA)

• Long, non-coding RNA molecules, (200nt – 100kb)
• Transcribed, primarily, by RNA Polymerase II
• Folded into highly-conserved secondary structures (tandem stem-loop structures)
• Estimated 20,000 - 100,000 InRNAs in the human genome, but the function of most IncRNAs is still unknown
• RNA secondary and tertiary structure is important

IncRNAs - similarities with mRNA

• Transcribed by RNA Polymerase II
• Processed after transcription, meaning they are Spliced, and 5'-capped and polyadenylated (3' -polyA tail)
• Expression regulated in tissue- and time-dependent manner - regulated by promoters/enhancers

IncRNAs - differences with mRNA

• Transcribed from various regions of DNA (introns, exons, intergenic regions)
• Shorter molecules
• Not translated
• Have Very low expression and greater tissue-specificity - Fewer transcription factor binding sites in promoters
• Primary sequence and secondary structure determines their function

Comparison of mRNA vs IncRNA

• mRNA:*
• Higher expression/Less tissue-specificity
• Exported to the cytoplasm
• Encode for protein
• Primary sequence determines their function (translation)
• IncRNA:*
• Lower expression/Greater tissue-specificity
• Both in the nucleus and cytoplasm
• No translation
• Primary sequence and secondary/tertiary structure determine their function
• mRNA – primary sequence determines binding of ribosomes and translation
• IncRNA function is determined by primary sequence and secondary/tertiary structure
• IncRNA interact with DNA by forming RNA-DNA triple helical structures
• IncRNA interacts with RNA (mRNA or miRNA) by base pairing (sequence-dependent)
• IncRNA interacts with proteins via stem-loop structures, creating protein binding sites
• IncRNAs change the expression of neighboring or distant genes by chromatin regulation, transcription regulation, and post-transcriptional regulation.

Chromatin regulation by IncRNA

• The guiding histone-modifying enzymes (e.g., histone acetyltransferases) to the gene promoter
• It acts as scaffolds to recruit protein complexes that modify histones

Regulation of transcription by IncRNA

• Recruiting transcription factors to gene promoters/enhancers - gene activation
• Acting as decoys by preventing transcription factor binding to gene promoters/enhancers - gene suppression
• IncRNA regulate mRNA in post-transcription, by editing via enzymes or by regulate RNA splicing by interacting with splicing factors
• mRNA translation is affected by IncRNA. IncRNA Compete with miRNA for binding to target mRNA which cause increased translation
• Each IncRNA regulates multiple mRNAs and miRNAs,
• IncRNAs regulate gene expression at multiple levels.
• Chromatin, transcription, post-transcription, miRNA
• IncRNAs regulate gene expression in a tissue- and time-dependent manner
• IncRNAs have multiple target genes, affecting cell division, cell differentiation, cell death, and metabolism
• Coordinates gene networks in homeostasis and normal development
• Abnormal changes in IncRNA levels lead to disease
• IncRNA are involved in Expression in specific tissues/times, and Knockdown, and are therefore important in embryonic development
• Dysregulated IncRNA expression in cancer can increase or decrease, causing abnormal cell function and disease