Regulatory RNAs Notes

Regulatory RNAs

Non-coding RNA (ncRNA)

  • ncRNA includes siRNA, miRNA, and piRNA.

Discovery of RNA Interference

  • Introduction of sense or antisense RNA into cells can interfere with gene function.

  • Double-stranded RNA is the best trigger for interference.

Small Regulatory RNAs

  • Three main classes: siRNA, miRNA, and piRNA.

siRNA

  • siRNAs are derived from double-stranded RNAs and are involved in cellular defense against exogenous RNAs (RNA interference).

  • Dicer chops up long dsRNAs into 20-25 base pair fragments, producing siR:siR* molecules.

  • Foreign RNAs are often double-stranded and are recognized and destroyed via RNA interference.

  • dsRNA is cleaved into small fragments by Dicer.

  • Fragments are loaded onto the RNA-induced silencing complex (RISC); the guide strand directs RISC to complementary full-length RNAs, which are cleaved by the Argonaute protein within RISC.

miRNA

  • miRNAs are encoded by specific genes and produced from hairpin structures by Dicer-like enzymes (Drosha in animals, Dcl1 in plants).

  • miRNAs begin as primary transcripts (pri-miRNAs); several miRNAs can be produced from the same pri-miRNA.

Enzymes Involved in RNA Processing

  • Drosha processes double-stranded primary RNAs into short precursors for Dicer processing.

  • Dicer processes double-stranded precursor RNA to 21-23 nucleotide RNAi molecules.

  • RNAse III family enzymes are involved in miRNA and siRNA processing.

miRNA and siRNA Similarities & Differences

  • miR:miR* and siR:siR* molecules both have 3′-OH and 5′ monophosphates and can undergo post-transcriptional modifications like methylation.

  • siR:siR* molecules are usually fully complementary, but miR:miR* molecules are usually not.

Heterochromatin Formation

  • MicroRNAs can promote heterochromatin formation.

  • RITS (RNA-induced transcriptional silencing) represses heterochromatin transcription using short single-stranded siRNA.

  • The RITS complex recruits a histone deacetylase and a histone methylase.

rasiRNAs

  • Repeat-associated siRNAs (rasiRNAs) help maintain centromeres in fission yeast.

  • Low-level bidirectional transcription of pericentromeric repeats produces dsRNA, which is cleaved by Dicer.

  • The RITS complex targets pericentromeric transcripts, recruiting an RNA-dependent RNA polymerase complex (RDRC).

Gene Silencing

  • miRNA functions can be modulated by ‘sponge’ RNAs.

piRNA Biogenesis

  • piRNAs are generated from long transcripts from specific loci (piRNA clusters) using a ‘ping-pong’ mechanism.

  • Transcripts are first processed by the PIWI domain of Piwi protein to yield primary piRNAs, which direct secondary processing.

  • piRNAs are generated from single-stranded piRNA precursor RNAs that are antisense to transposon mRNAs.

  • Zucchini nuclease cleaves the initial transcript into fragments loaded onto Piwi proteins.

  • Aubergine (Aub)-loaded piRNA directs cleavage of the transcript, generating another piRNA.

Long Intergenic Non-Coding RNAs (lincRNAs)

  • Transcription of non-protein-coding regions can produce long transcripts.

  • Example: 17000-nucleotide Xist RNA involved in X chromosome inactivation.

  • The processed transcript coats the X chromosome and recruits the Polycomb protein complex.

RNA-Protein Interactions

  • Proteins have specific RNA-binding motifs.

  • The most common is the RNA-recognition motif (RRM).

  • KH and PAZ domains also bind single-stranded RNA.

  • RNase III family enzymes have double-stranded RNA-binding domains.

Argonaute Proteins

  • Eukaryotic sRNAs carry out functions in association with Argonaute proteins.

  • Argonaute proteins have four domains: N, PAZ, Mid, and PIWI.

  • PAZ binds the 3′ end of bound RNA, Mid interacts with the 5′ end, and PIWI interacts with the whole RNA.

  • miRNA and siRNA molecules are loaded onto Argonaute proteins.

  • Single-stranded piRNAs are loaded directly onto Piwi proteins.

RISC Complex

  • The guide RNA-Argonaute complex is called the RISC complex (RNAi-induced silencing complex).

  • miRISC usually pairs initially via a 2-8 nucleotide ‘seed’ sequence, while siRISC can bind along its full length.

  • miRISC binding causes translational repression; siRISC activates the PIWI domain.

Eukaryotic sRNA Classes

  • MicroRNAs (miRNAs): derived from primary transcripts and generally downregulate cytoplasmic