20.01.11 Regulatory noncoding RNAs Flashcards

1
Q

What are ncRNAs

A
  • Noncoding RNAs
  • Constitute the majority of the transcribed human genome (60%)
  • Not translated into protein
  • Regulators of gene expression, modulating cell proliferation, apoptosis, cell cycle, DNA methylation.
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2
Q

Categories of ncRNAs

A
  • Long non-coding RNAs (lncRNAs)- >200nt
  • Small noncoding RNAs (sncRNAs) <200nt
    a) microRNAs (miRNA)
    b) piwiRNAs (piRNA)
  • Endogenous small interfering RNAs
  • circular RNA (circRNA)
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3
Q

What are microRNAs (miRNA)

A
  • Small ncRNAs.
  • 22 nt long. Highly conserved in eukaryotic organisms
  • Regulate gene expression by post-transcriptional gene silencing
  • Encoded by introns of non-coding or coding transcripts
  • 70% are polycistronic transcription units (co-expressed)
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4
Q

How do miRNAs function

A
  • Bind complementary sequences within the 3’UTR of target mRNA
    a) Inhibit translation by preventing binding of the translational machinery
    b) Promote mRNA degradation (by deadenylation of polyA tail)
  • Nt 2-7 (from 5’ end) is highly conserved, seed region. Recognises target mRNA.
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5
Q

How are miRNAs produced

A
  • RNA polymerase II transcribes long primary miRNA transcripts (1kb).
  • Precursor is processed in the nucleus, cropped into 65nt pre-miRNAs. Enzymes include DGCR8 (DiGeorge critical region gene 8). Deletions of DGCR8 are implicated in DiGeorge syndrome
  • pre-miRNAs translocate to cytoplasm. Cleaved to produce miRNA duplexes.
  • duplex RNA is loaded into the RISC(RNA induced silencing complex)/Argonaute complex. Helix is unwound and passenger stand it degraded.
  • RISC directs regulation of mRNA.
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6
Q

miRNAs in disease (autosomal dominant)

A
  • Hereditary progressive hearing loss. Loss of seed region of miR-96. Interferes with pre-miRNA processing.
  • Hereditary keratoconus with cataracts. Mutations in seed region of miR-184. Mutant form can’t compete for sites on INPPL1 and ITGB4 genes. mi-184 highly expressed in cornea and lens.
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7
Q

Involvement of miRNA in cancer

A
  • miRNA disruption
  • Disruption of miRNA-target gene regulation
  • inhibition of global miRNA processing
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8
Q

miRNA in cancer, examples

A
  • Dysregulation of miR-21, overexpressed in a variety of cancers including lung and colorectal. Higher expression linked with metastases. Possible diagnostic marker.
  • Chromosome 13q14 deletion. Causes B cell chronic lymphocyte leukaemias. Due to dysregulation of miR-15 and 16.
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9
Q

What are small interfering RNA (siRNA or endo-siRNA)

A
  • small double-stranded ncRNA 21-22nt long
  • Produced as first stage in RNA interference.
  • siRNA precursors include: exogenous dsRNA, shRNA, centromeres, transposons.
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10
Q

How are siRNAs produced

A

-Dicer catalyses the production of siRNAs from the degradation of long double-stranded RNA and from the processing of small hairpin RNAs (shRNA).

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11
Q

What is RNA interference (RNAi)

A

Cellular defence mechanism triggered by the presence of dsRNA and shRNA resulting in degradation of specific gene transcripts.

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12
Q

Steps of RNAi

A
  1. Long dsRNAs and shRNAs processed in cytoplasm by RNase DICER into siRNAs
  2. siRNAs taken up by Argonaute subunit of RISC (RNA-induced silencing complex)
  3. Duplex siRNA is unwound and passenger strand dissociates
  4. Antisense RNA strand guides siRISC to complementary target mRNA. Ago2 has endonucleolytic activity so results in mRNA cleavage.
  5. siRNA-loaded RISC is recycled for several rounds of mRNA cleavage.
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13
Q

What are Piwi interacting RNAs (piRNA)

A
  • Small ncRNAs. 23-36nt long
  • Function with AGO and PIWI proteins to regulate transposon activity and chromatin states
  • piRNAs expressed in adult differentiated cells
  • Piwi-class of Argonaute proteins are expressed only in germline cells.
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14
Q

How are piRNAs formed

A

-From a single strand precursor (up to 200kb) in a Dicer independent mechanism.

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15
Q

What 3 groups of piRNAs are there

A
  • Transposon derived (made from both strands so sense and antisense)
  • mRNA derived (in 3’ UTR of mRNA from which the originate- sense strand)
  • lncRNA derived
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16
Q

How does piRNAs recognise target

A

By associating with PIWI proteins to create piRISC (piRNA-induced silencing complex)

17
Q

piRNAs in disease

A
  • LOF mutations in piRNA derepress transposons, leads to them inserting copies elsewhere in genome. Activates Chk2 DNA damaging checkpoint. Leads to defects of microtubule organisation during gonadal development, leading to infertility.
  • piR-651 is upregulated in gastric, colon and lung cancer. Inhibition of piR-651 arrests cells in G2/M phase
18
Q

What are long ncRNAs (lncRNAs)

A
  • ncRNAs over 200nt long

- Similar to mRNAs. Transcribed by RNA pol III, may be polyadenylated, can show complex splicing patterns.

19
Q

Main types of lncRNAs

A
  • Antisense (overlap known protein coding genes)
  • Intronic (encoded in the introns of coding genes)
  • Overlapping transcripts (overlap protein coding genes)
  • Intergenic (encoded completely within intergenic genomic spaces between protein coding loci)
20
Q

Actions of lncRNAs

A
  • Transcription factor inactivation
  • Transcriptional regulation
  • Transcription-mediated silencing
  • Post transcriptional regulation
  • Modification of alternative splicing
  • Regulation of mRNA stability
21
Q

Examples of lncRNAs with posttranscriptional regulation activities

A
  • Xist lncRNA in females. Induces X inactivation.

- HOTAIR regulates gene expression by associating with chromatin-modifying complexes

22
Q

lncRNAs in disease

A
  • Many imprinted gene loci express lncRNAs, which have roles in reuglation of neighbouring imprinted genes in cis.
  • Air: monoallelically expressed from the paternal allele associates with histone methyltransferase and localise to chromatin to silence 3 imprinted genes. lncRNA H19 involved in imprinting at 11p15 imprinted cluster, associated with Beckwith Wiedemann syndrome.
23
Q

What are circular RNAs (circRNA)

A

-Circular (close loop structure) single stranded RNA molecules
Without polarity or polyadenylated tail. Resistant to RNAse R digestion and more stable than linear RNA molecules
-Abundant
-Generated during splicing of mRNA by the spliceosome, at the expense of the canonical mRNA. Therefore a regulator of mRNA production.

24
Q

What is the competing endogenous RNA hypothesis

A

competing endogenous RNAs (abbreviated ceRNAs) regulate other RNA transcripts by competing for shared microRNAs (miRNAs)

25
Q

Uses of ncRNAs as diagnostic/therapeutic tools

A
  • ncRNAs are aberrantly expressed in different conditions. Could be useful as diagnostic/ prognostic markers
  • Therapeutic agents to silence defective alleles, modulate alternative splicing to correct defective gene expression.
26
Q

Examples of ncRNAs in clinical trials

A
  • MRX34- mimics tumour suppressor miRNA miR-34, which is often loss in solid or hematological malignancies. Induces cell cycle arrest, senescence and apoptosis, via p53 pathway.
  • ASOs such as Spinraza to modify pre-mRNA splicing of SMN2 to promote exon 7 inclusion.