Lecture 13 & 14 Notes

Non-protein coding RNAs regulate gene expression at different levels.
- Long noncoding RNAs: Act at the DNA level, influencing chromatin structure and gene transcription.
- siRNAs, microRNAs, short hairpin RNAs: Act directly at the RNA level, modulating mRNA stability and translation.

Target transposable element products at the RNA level, specifically during the RNA intermediate stage to prevent transposition.
Interact with a unique set of Piwi proteins, functionally analogous to the RISC complex but specific for transposon silencing.
Hybridize to transposable element transcripts; along with Piwi enzymes, they guide the degradation of these transcripts at the RNA level.
Silence retrotransposons by directing DNA methylation and heterochromatin formation at their genomic loci.
Exhibit varying lengths, typically 24-32 nucleotides, which is a key characteristic for distinguishing them from other small regulatory RNAs.

Synthesized from a longer RNA molecule that folds into a stem-loop structure, a characteristic hairpin shape.
The primary transcript is processed to release a microRNA precursor.
A microRNA is selected from this long RNA precursor through several enzymatic steps.
Synthesized in the nucleus, cleaved by Drosha, and then translocated from the nucleus to the cytosol via exportin-5.
Further processed in the cytoplasm by the enzyme Dicer, which cuts the stem-loop to produce a short, double-stranded RNA.
One strand of the duplex is selected as the mature microRNA and integrates with Argonaut proteins to form the functional RISC complex.
The RNA component provides specificity, enabling the RISC complex to hybridize with a particular mRNA/RNA target based on sequence complementarity.
The RISC complex, guided by the microRNA, results in:
- Slicing/degradation of the targeted mRNA, if there is a high degree of complementarity.
- Inhibition of translation by blocking the ribosome from translating mRNA into protein, when complementarity is partial.
P bodies: Cytoplasmic granules where mRNA degradation and storage occur, serving as hubs for post-transcriptional gene regulation.
MicroRNA and Argonaut proteins combine to form the RISC complex within these P bodies.

Used to visualize the location and distribution of proteins within cells, demonstrating regions where P bodies are concentrated.
Green fluorescence indicating the presence of decapping proteins, which are involved in mRNA degradation.
Argonaut proteins, a core component of the RISC complex, also visualized.
Yellow regions, representing the overlap between decapping and Argonaut proteins, confirm the location of P bodies.

Synthesized from long double-stranded RNA molecules, which can be introduced exogenously or produced from endogenous sources.
The RNA is processed into short, double-stranded fragments, typically 21-23 nucleotides long, by the enzyme Dicer.
These RNAs do not undergo splicing, ensuring they maintain a direct representation of the targeted sequence.
Can be encoded by an exon or intronic region, allowing for precise targeting of specific gene transcripts.
The double-stranded RNA is cleaved to the appropriate size by Dicer, generating siRNA duplexes.
One strand, known as the guide strand, is selected and complexes with Argonaut proteins, along with the rest of the RISC proteins.
Can target the mRNA/RNA of interest with high specificity, leading to its degradation mediated by the RISC complex.
Can also interact with RNA that is being synthesized, interfering with transcription and targeting the nascent transcript for degradation.
Can recruit histone methylation of the DNA, leading to transcriptional silencing through epigenetic modifications.
The coding region for this RNA can be methylated, further reinforcing transcriptional repression.
RITS Complex: A complex involved in transcriptional repression, where the transcript is not fully synthesized, often mediated by siRNAs.

RITS (RNA-induced initiation of transcriptional silencing): Predominantly associated with siRNAs and involved in establishing heterochromatin.
RISC (RNA-interfering sensing complex): Can be formed with either siRNA or microRNA, mediating mRNA cleavage or translational repression.