Lecture 11: Small RNA Biogenesis

State what distinguishes RNA from DNA in sugar, base and strandedness

Here are the answers to the learning objectives for the Biogenesis of Non-coding Regulatory RNAs lecture based directly on your provided lecture slides:

1. Distinctions Between RNA and DNA

• Sugar: RNA contains ribose, which has a hydroxyl group (OH) at the C2 position, whereas DNA contains deoxyribose, which has a hydrogen atom (H) at C2

• Base: RNA uses uracil (U) instead of thymine (T), alongside adenine (A), guanine (G), and cytosine (C).

• Strandedness: RNA is single-stranded, whereas DNA is typically double-stranded

Define a non-coding RNA, give the size that separates long from small non-coding RNAs, and say how lncRNAs are classified by genomic context

• Definition of non-coding RNA (ncRNA): RNAs that do not directly code for polypeptides.

• Size Threshold: 200nt divides long non-coding RNAs (200nt) from small non-coding RNAs (20–30 nt).

• Classification of lncRNAs by Genomic Context:

  • Stand-alone transcription units

  • Enhancer-associated (eRNAs) or promoter-associated (e.g., TSSa-RNAs, uaRNAs, pasRNAs, PROMPTs)

  • Intronic (transcribed within introns of other genes)

  • Pseudogenic

    Antisense (NATs), which can be divergent (no overlap), terminal (partial overlap), or nested (complete overlap)

  • Small RNA-containing (hosting small RNAs like hairpins within their unit)

Give the three principles that hold for most regulatory RNAs

• Primary transcripts of regulatory RNA molecules are often processed to yield the final functional molecule.

• Regulatory RNAs usually employ base pairing with their RNA and DNA targets.

• Regulatory RNAs often interact with other components (such as proteins) to carry out their functions.

Describe the three ways base pairing by a regulatory RNA can change what happens to its target

• Disruption/blocking of protein binding to the target RNA.

• Alteration/change of RNA structure.

• Recruitment/tethering of proteins to the site.

Explain what Dicer does, and how its structure sets the size of the product

• Function: Dicer is a Class III RNase III family enzyme that cleaves long double-stranded RNA (dsRNA) or hairpin-structured pre-miRNAs into short RNA duplexes ($\sim 21–25\text{ nt}$).

• How Structure Sets Product Size: Dicer acts like a ruler; its PAZ domain anchors the $3'$ end of the RNA stem, and the distance from the PAZ domain to its dimeric RNase III catalytic domain determines the specific length of the cleaved small RNA product.

State what Argonaute contributes to silencing, and what happens to the passenger strand

• Argonaute Contribution: Argonaute (AGO) proteins bind the mature small RNA guide strand and form the core of the RNA-induced silencing complex (RISC), using the guide to direct target RNA degradation, translational block, or transcriptional gene silencing (heterochromatin formation).

• Fate of the Passenger Strand:

  • The strand with the less stably base-paired 5' end is retained as the guide strand.

  • The passenger strand (miR^* or siR^*) is discarded and degraded. For siRNAs, the endonuclease (PIWI) domain of Argonaute cleaves the passenger strand to facilitate its unwinding and release.

Trace the miRNA pathway from primary transcript to loaded silencing complex, and say which steps are nuclear and which are cytoplasmic

• Transcription: Primary microRNA (pri-miRNA) stem-loop structures are transcribed by RNA Polymerase II or III.

• Primary Cleavage: In animals, the Microprocessor complex (Drosha + DGCR8/Pasha) cleaves the 5' and 3' extensions of the pri-miRNA to release a ~60–70nt hairpin precursor (pre-miRNA). (In plants, DCL1 performs this step in the nucleus).

• Nuclear Export: Pre-miRNA is exported out of the nucleus into the cytoplasm via Exportin-5 / Ran-GTP.trans-acting siRNAs (tasiRNAs)

  • Why Secondary: Their biogenesis is triggered by a primary small RNA (miRNA) through AGO-mediated cleavage of a long single-stranded precursor transcript derived from $TAS$ loci.

  • Amplification Mechanism: After initial miRNA cleavage, RNA-dependent RNA Polymerase 6 (RDR6) converts the cleaved single-stranded $TAS$ transcript into double-stranded RNA, which DCL4 then chops into phased $21\text{ nt}$ tasiRNAs to amplify target silencing across non-identical mRNAs.

  Piwi-interacting RNAs (piRNAs)

  • Why Secondary: Generated via the "ping-pong" amplification loop where primary piRNAs drive the cleavage and generation of reciprocal secondary piRNAs.

  • Amplification Mechanism: A primary piRNA loaded into a Piwi protein cleaves a transposon target transcript; the cleavage product is then trimmed and loaded into a second Piwi protein to form a secondary piRNA, which in turn targets and cleaves the original primary transcript, establishing an amplifying feed-forward cycle.

Contrast miRNA and siRNA biogenesis by the origin of the precursor and the enzymes required

Name the enzymes in these pathways that plants and animals do not share

• Animals have (Plants lack): Drosha and DGCR8 (Pasha) (used for nuclear pri-miRNA cleavage; plants use DCL1 instead).

• Plants have (Animals lack): RNA-dependent RNA Polymerases (RDRs) (such as RDR2 and RDR6, used to synthesize complementary RNA strands from single-stranded templates), specialized RNA polymerases Pol IV and Pol V, and Dicer-Like (DCL) proteins

Say what makes tasiRNAs and piRNAs secondary small RNAs, and how each amplifies silencing

trans-acting siRNAs (tasiRNAs)

• Why Secondary: Their biogenesis is triggered by a primary small RNA (miRNA) through AGO-mediated cleavage of a long single-stranded precursor transcript derived from TAS loci.

• Amplification Mechanism: After initial miRNA cleavage, RNA-dependent RNA Polymerase 6 (RDR6) converts the cleaved single-stranded $TAS$ transcript into double-stranded RNA, which DCL4 then chops into phased 21nt tasiRNAs to amplify target silencing across non-identical mRNAs.

Piwi-interacting RNAs (piRNAs)

• Why Secondary: Generated via the "ping-pong" amplification loop where primary piRNAs drive the cleavage and generation of reciprocal secondary piRNAs.

• Amplification Mechanism: A primary piRNA loaded into a Piwi protein cleaves a transposon target transcript; the cleavage product is then trimmed and loaded into a second Piwi protein to form a secondary piRNA, which in turn targets and cleaves the original primary transcript, establishing an amplifying feed-forward cycle.