Main types of RNA: mRNA, tRNA, rRNA

Other types of RNA: shRNA, sRNA, ncRNA, miRNA, snRNA, circRNA, gRNA

These roles are diverse and involved in protein synthesis and RNA splicing

Gene expression and cell defense is regulated with ncRNAs

ncRNA is 20x more prominent than mRNA in most cells and is ID’d by bulk RNA sequencing

IncRNA: long noncoding RNA

Inc RNA has many roles in eukaryotic chromatin structure and regulation

Prokaryotic sRNA has various mechanisms in bacteria

• sRNA can bind mRNA directly as antisense to block translation

• snRNA can bind mRNA to change and activate transcription

• snRNA can bind repressor proteins to recruit promoters to block transcription

One role of IncRNA is maintaining genomic intergrity

• Types of IncRNA: TERC-RNA and piRNA(Piwi-interacting RNA)

• TERC maintains telemeres

• piRNA are encoded in mammalian cells and silences transposable elements to prevent jumping

microRNAs (miRNA): bind to complementary sequence of target mRNAs

miRNAs play roles in gene silencing, cell cycle regulation, cancers

small interfering RNAs (siRNA): RNAi pathways, form RISC, and cleaves complementary mRNAs

siRNAs are important for viral defense and can be a tool for gene silencing

PIWI-interacting RNAs (piRNA): interact with PIWI proteins to silence and regulate transposons to maintain genomic intergrity

long noncoding RNAs (IncRNAs): important for transcription regulation and chromatin remodelling

Small nucleocular RNAs (snoRNAs): guide chemical modifications through methylation of rRNA, tRNA, and snRNA

Small nuclear RNAs (snRNAs): components of the splicesomes (splices pre-mRNA) as well as RNA processing

Transfer RNAs (tRNAs): carry amino acids to ribosome and decodes mRNA into proteins during translation

Ribosomal RNA (rRNAs): make up the core structure of ribosomes through protein synthesis

Circular RNA (circRNAs): important for transcriptional regulation and can act as microRNA sponges which impacts miRNA availability and gene expression

X-inactive specific transcript (Xist): chromosomal structure of female mammals

Telomerase RNA component (TERC): replicates telomeres which improves chromosomal stability, as well as modulating chromatin

guideRNA (gRNA): used in CRISPR, gene editing via the gRNA-Cas9 complex which searches for and binds to target DNA

Compare and contrast siRNA and miRNA

• Similarity: same length (20-25 nucleotides), both used in the RNAi mechanisms, use DICER to process per-miRNA and double stranded siRNAs, both loaded onto RISC, both reduce gene expression through target degradation

• Differences:

  • siRNA is from exogenous dsRNA while miRNA is endogenous and transcribed from genes

  • siRNA defends against foreign RNA while miRNA regulates gene expression during development

  • siRNA targets complementary mRNA and then cleaves or degrades it, while miRNA is only partially complementary with mRNA, leading to translation repression or degradation

  • miRNA has a hairpin loop from single stranded RNAs folding on itself

  • siRNA has a single mRNA target while miRNA has multiple mRNA targets and imperfectly binds

  • siRNA os used for targeted gene silencing for specific disease genes while miRNA can be used for cancers due to multiple gene targets

Delivery modes of siRNA in mammalians

• Chemically synthesized siRNAs (which are shorter double stranded RNAs) act as endogenously produced siRNAs)

• Methyl additionals to 2”-OH ribose make the mRNA target more accessible to siRNAs

• Liposome delivery: protects siRNAs from degradation by must escape the endosome to release the siRNA into the cytoplasm to engage in RISC. It is easy to formulate with targeting ligands but is inefficient, can have off target effects, and has a short circulation time meaning it is rapidly cleared

• Transfection delivery: lipids or polymers which are positively charged to help siRNA which is negatively charged. It is highly efficient and reagents are easily available. It can be applied to high-throughput screening. However, the reagents can be toxic, siRNAs are transient and are not stable

• Microinjection delivery: a micropipette or needle is used to introduce the siRNA into specific cells. It is precise, efficient, and no carrier is required to avoid toxicity. However, it is invasive, labor intensive, it is not scaleable, and can potentially damage cells

circRNAs are a type of IncRNA and contains binding sites for many miRNAs

circRNAs act as a sponge or inhibitor for miRNA function in vivo, and is more stable than linear RNAs

circRNA contains sequester miRNAs to prevent interaction with target mRNAs and indirectly regulates gene expression