Flashcards in 18.01.11 Regulatory non-coding RNA Deck (48)
Give three examples of regulatory non-coding RNA species.
What is the typical structure of a miRNA?
Small, naturally occurring ncRNAs ~21-22 nt long, highly conserved in eukaryotic organisms.
What is the role of miRNAs?
Regulate gene expression by post-transcriptional gene silencing (>60% protein coding genes)
Some regulate specific individual targets
Others are master regulators of a process (regulate expression of 100’s genes simultaneously)
Some regulate targets cooperatively.
Involved in regulating many processes e.g. proliferation, differentiation, apoptosis and development
How many miRNA molecules have been identified in humans?
In humans, 1881 precursors (pre-miRNAs) and over 25,000 mature miRNA molecules have now been identified (). A recent study identified a further 3,700 new miRNA many with tissue specific expression (Londin E (2015) Proc Natl Acad Sci U S A. 112: E1106-15.).
How do miRNAs enact their function?
Function by binding complementary sequences within the 3’ UTR of target mRNA then
(1) Inhibiting translation by preventing binding of the translation machinery or
(2) Promote mRNA degradation (e.g. through deadenylation of the PolyA tail)
Where are the majority of miRNAs encoded?
Majority encoded by introns of non-coding or coding transcripts, some found in exonic regions. Many miRNAs are known to reside in introns of their pre-mRNA host genes and are transcribed in the same direction as the pre-mRNA – leading to hypothesis that these miRNAs share their regulatory elements.
~70% of human miRNAs are found in defined poly-cistronic transcription units– generally co-transcribed - with a large proportion located in sense orientation within the introns of protein coding genes.
What is the seed region of miRNA?
In mature miRNAs there is strong conservation of nt 2-7 (from 5’ end), known as the seed region (crucial for recognition of target mRNA).
miRNAs with identical sequences at nt 2-8 belong to same ‘miRNA family’. miRNAs in a cluster are often related to each other suggesting that clusters may have arisen by gene duplication.
How can mature miRNAs function as developmental regulators?
Due to transcription of the miRNA genes by RNA polymerase II, mature miRNAs can function as developmental regulators by blocking expression of specific genes in specific tissues, and at specific times.
Where does miRNA maturation occur?
Unlike mRNAs, miRNA maturation begins in the nucleus and finishes in the cytoplasm.
Where are miRNA response elements found?
miRNA response elements (MREs) are found in coding and non-coding RNA. Other non-coding RNA species have roles in regulating (and being regulated by) miRNAs that bind to their MREs (eg CDR1as and miR-7, see Circular RNA section below).
What is the pathway involved in the regulation of mRNA by miRNAs?
Pathway by which miRNAs regulate mRNA translation involves mechanism used by cells as a defence against exogenous mRNA e.g. viral infection called: Post Transcriptional Gene Silencing (PTGS pathway)
How are miRNA molecules formed?
miRNA genes transcribed by RNA pol II into long primary miRNA (pri-miRNA) transcripts (typically over 1 kb) contains stem loop structure, a 5’ cap & a polyA tail (similar to mRNAs).
Maturation initiated by the RNASEN/ Drosher microprocessor complex - contains DGCR8 essential cofactor (RNA binding activity) and Drosher (RNAse III type endonuclease). Drosher crops the stem loop structure from pri-miRNA to produce ~65nts length pre-miRNA within the nucleus. Deletion of DGCR8 gene implicated in DiGeorge syndrome.
Hairpin precursor miRNAs (pre-miRNAs) are transported to the cytoplasm by exportin 5 (XPO5)
The Dicer-TARBP2 complex (RNAse III type endonuclease Dicer and argonaute protein TAR RNA-binding protein 2) removes the loop region from pre-miRNAs to create an miRNA duplex.
Duplex RNA is loaded into the RNA induced Silencing Complex (RISC)/ Argonaute complex, the helix is unwound and the passenger strand is degraded.
RISC directs regulation of mRNA by recognizing and binding to the complementary sequences on the targeted mRNA transcript. If the pairing is 100% complementary between miRNA and mRNA the mRNA will be degraded, if only partially complementary this results in translational inhibition of the mRNA.
Give two examples of AD genetic disorders associated with miRNA mutations.
1. Hereditary progressive hearing loss due to a heterozygous mutation in the seed region of miR-96 (Mencia et al 2009 Nat Genet 41: 609-613), and a mutation in miR-96 outside of the seed region that hinders precursor processing, probably by interfering with Dicer cleavage (Solda et al 2012 Hum Mol Genet 21: 577-585).
2. Hereditary keratoconus with cataracts due to a heterozygous mutation in the seed region of miR-184 (Hughes et al 2011 Am J Hum Genet 89: 628-33). The mutant form fails to compete with miR-205 for overlapping target sites on the 3' UTRs of INPPL1 and ITGB4 genes. Although these target genes and miR-205 are expressed widely, the phenotype is restricted to the cornea and lens because of the very high expression of miR-184 in these tissues.
3. Germline hemizygous microdeletions at 13q31.3 encompassing the MIR17HG gene result in a syndrome of microcephaly, short stature and digit abnormalities (de Pontual et al 2011 Nat Genet 43: 1026-30). The MIR17HG locus encodes for miR-17-92, a polycistronic miRNA cluster from which six distinct miRNAs are produced. Mutations in the Human MYCN transcription factor result in a similar phenotype likely due in part to MYCN’s regulation of MIR17HG gene expression.
4. Mutations affecting miRNA processing machinery
- Mutations in Drosha microprocessor complexes Fus and TDP-43 are responsible for ~50% of familial ALS
- Mutations in RISC complex component Fragile X mental retardation 1 protein (FMRP) cause fragile X syndrome.
How are miRNAs involved in cancer?
miRNAs involved in Cancer via:
1) Direct disruption of miRNA loci
2) Disruption miRNA-target genes regulation
3) Inhibition of global miRNA processing.
Give examples of how miRNA can be involved in cancer.
miR15 and miR16 dysregulation in most B cell chronic lymphocytic leukaemias as a result of chromosome 13q14 deletion.
Dysregulation of the miR-21 identified as one of the most consistently overexpressed miRNA in a variety of different cancers including lung and colorectal cancer. Controls a wide range of biological processes and has role in carcinogenesis, recurrence, metastasis and resistance. Could be used as a diagnostic and prognostic marker of malignant tumours and may promote radioresistance of cancers.
Higher expression of miR-21 in serum of lung cancer patients is associated with lymph node mestatases and lymph node staging – could be used as a diagnostic marker for early stage lung cancer. Overexpression of miR-21 decreases the sensitivity of Gastric Cancer cells to Trastuzumab, significantly suppresses Trastuzumab induced apoptosis.
Which miRNA is associated with Li-Fraumeni syndrome?
The G allele of a polymorphism (rs2043556) within the miRNA miR-605 has been shown to accelerate the age of on-set of Li-Fraumeni syndrome by 10 years. May help to explain some of the intra-family heterogeneity of symptoms in LPS. The G allele appears to cause a defect in the processing efficiency of its host miRNA thus reducing expression of the mature miRNA, leading to dysregulation of MDM2, higher expression of which has been associated with younger ages of onset
What is the structure of siRNAs?
Small, double stranded ncRNA molecules ~21-22nt long
Endogenous short interfering RNAs arise as a result of production of limited amount of natural double-stranded RNA in the cell and occasional transcription of some pseudogenes.
How are siRNAs formed?
The enzyme catalyzes the production of siRNAs from the degradation of long double-stranded RNA (dsRNA) and from the processing of small hairpin RNA (shRNA).
What are the precursors of siRNAs?
siRNA precursors include: exogenous dsRNA and shRNA, centromeres, transposons and other repetitive sequences, convergent mRNA transcripts and other natural sense-antisense pairs, duplexes involving pseudogene-derived antisense transcripts and the sense mRNAs from their cognate genes.
How can siRNAs mediate transcriptional gene silencing?
siRNAs can mediate transcriptional gene silencing by direct heterochromatin formatio
What is RNA interference (RNAi)?
a cellular defence mechanism triggered by the presence of dsRNA and shRNA resulting in degradation of specific gene transcripts (hence potent and long lasting silencing of expression).
What is the role of siRNAs in RNAi?
siRNA produced as the first stage in the process of RNA interference
long dsRNAs and shRNAs are processed in the cytoplasm by the RNase Dicer into siRNAs.
The resulting siRNAs containing the characteristic 19-nt duplexed region with 2-nt 3’ overhangs and 5’-terminal phosphate groups are taken up by the Argonaute multi-subunit RNA-induced silencing complex (RISC).
The duplexed siRNA bound to the Ago2 protein, the central component of siRISC, is unwound and the passenger strand rapidly dissociates.
Once unwound, the antisense RNA strand guides siRISC to the complementary site in the target mRNA which engages the endonucleolytic (slicer) activity of Ago2, resulting in mRNA cleavage.
The siRNA-loaded RISC is recycled for several rounds of mRNA cleavage.
What is the stucture of piwiRNA?
Small ncRNAs ~23-36 nt long
Most diverse class of ncRNA molecules; over 15,000 human piRNAs have been identified.
What is the function of piwiRNAs?
Function with AGO and PIWI proteins to regulate transposon activity and chromatin states. PIWI proteins expressed mainly in germline but piRNAs expressed in many adult differentiated cells.
Role: Limit transposition by retrotransposons (LINE-1) in mammalian germ cells.
piRNAs associate with PIWI proteins to create an active piRNA-induced silencing complex (piRISC) that recognises complementary targets.
piRNAs recently reported to play an important role in the control of gene expression – can share post-transcriptional activities by interacting with other Argonaute proteins and silencing mRNAs in cytoplasm
How do piwiRNAs differ from miRNAs and siRNAs?
Differ from miRNA/siRNA: they are longer, produced from single strand precursor up to 200kb (Dicer-independent mechanism), associate with the Piwi-class Argonaute proteins, expressed only in germline cells.
Where in the genome are piwiRNAs located?
89 large clusters distributed across the genome; individual clusters span 10 kb - 75 kb with an average of 170 piRNAs per cluster. Cleaved from large multigenic transcripts. (
What are the three groups of piwiRNAs?
(1) transposon derived - transcribed from both strands to produce sense and antisense piRNAs,
(2) mRNA derived piRNAs – found in 3’UTR of mRNA from which they originate (sense strand),
(3) lncRNA-derived piRNAs – produce piRNAs from entire transcript.
Give disease examples associated with piwiRNAs.
piRNA and infertility - Loss of function mutations in piRNAs derepress transposons allowing them to insert copies of themselves or relocate within the genome - activates Chk2 DNA damage checkpoint resulting in defects of microtubule organization and axis specification during gonadal development, which often lead to infertility.
piRNA and cancer - potential biomarker in human cancer e.g. piR-651 upregulated in gastric, colon, lung and breast cancer and in several cell lines. Growth of gastric cancer cells inhibited by piR-651 inhibition – arrest in G2/M phase.
piR-823 expression lower in gastric cancer tissues. Increasing the level of piR-823 inhibits cell growth
What is the structure of long ncRNAs (lncRNA)?
ncRNAs over 200 nt long
~20, 000 lncRNA transcripts have been identified; Little evolutionary conservation of lncRNA between species ie only small regions of lncRNA are functional.
Many similarities to mRNA: Often transcribed by RNA Pol II, may be polyadenylated, can show complex splicing patterns
What different types of lncRNAs exist?
Several types: antisense lncRNAs (that overlap known protein coding genes), intronic lncRNAs (that are encoded within introns of protein coding genes), overlapping transcripts lncRNA (that overlap protein coding genes) and intergenic lncRNAs (that are encoded completely within intergenic genomic space between protein coding loci).
What is the role of lncRNAs?
lncRNAs affect many biological processes e.g. regulation of gene expression both in cis and in trans, guidance of chromatin-modifying complexes, X chromosome inactivation (Xi) and genomic imprinting, nuclear compartmentalization, nuclear-cytoplasmic trafficking, RNA splicing and translational control.
Some are known to be tissue specific.
What are the main actions of lncRNAs.
1. Transcription factor inactivation
2. Transcriptional regulation
3. Transcription-mediated silencing
4. Post-transcriptional regulation
5. Modification of alternative splicing
6. Translational control or by regulation of mRNA stability.
Give examples of lncRNA and human disease.
Dysregulation of lncRNAs appears to be a primary feature of many complex human diseases, including leukaemia, colon, prostate and breast cancer, Alzheimer’s disease and spinocerebellar ataxia type 8. (recent reviews: Colorectal cancer- Shuwen et al 2018, Wound healing- Luan et al 2018, Cheetham et al 2013). For examples of lncRNA in cancer progression see figure C.
The antisense RNA, BACE1-AS is upregulated in patients with Alzheimer's disease leading to increased concentrations of beta amyloid, the main constituent of senile plaques. BACE1-AS concentrations are elevated in subjects with Alzheimer's disease and in amyloid precursor protein transgenic mice.
lncRNA and genomic imprinting - Many imprinted gene loci express lncRNAs that appear to play major roles in regulating the expression of neighboring imprinted protein-coding genes in cis e.g. Air: monoallelically expressed from the paternal allele, associates with a histone methyltransferase and localizes to chromatin to silence three imprinted genes. The lncRNA H19 is involved in imprinting at the 11p15 imprinted cluster associated with Beckwith–Wiedemann syndrome.
What is the structure of circular RNA?
Circular single stranded RNA molecules without polarity or polyadenylated tail. Resistant to digestion by RNase R and therefore more stable than linear RNA molecules.
Previously assumed to be splicing errors but now beginning to be recognised as a further layer of transcriptional regulation
How are circular RNA molecules formed?
Mostly generated during splicing of messenger RNA by the spliceosome. ‘Back-splicing’ reactions covalently link the 3’ end of an exon to the 5’ end of an upstream exon. Promoted by the binding of intronic reverse complementary sequences flanking the exons. (See figure F)
Exons that generate circRNAs are typically not alternatively spliced. Thus, circRNAs are typically generated at the expense of canonical mRNA iso-forms, which can be much less abundant than the circRNAs they host. This suggests that circRNA biogenesis per se might be an important regulator of mRNA production.
How are circular RNA molecules regulated/act as regulators?
RNA binding splicing factors such as Quaking protein and musclebind have been shown to regulate circRNA biosynthesis. Recognition sequences for Quaking protein in flanking introns sufficient to promote circRNA from exons.
Can serve as transcriptional regulators or sponges for miRNAs. Eg CDR1as which is enriched in the brain and has >70 binding sites for miR-7. miR-7 titers away CDR1as (ciRS-7) and promotes its compartmentalisation in the P bodies. miR617 binding to CDR1as results in degradation. (Taulli et al 2013)
What is the association between circRNA and human disease.
No clear role as yet but changes in expression of circRNAs have been shown during epithelial-mesenchymal transition (EMT). Epithelial cell cancer development requires EMT to become invasive and to metastasise.
How can ncRNAs be used as diagnostic tools?
ncRNAs are aberrantly expressed in different conditions such as cancer, cardiovascular disease, etc and therefore may be used as diagnostic/prognostic markers:
early diagnosis of cancers difficult to detect (ex: colon) may be possible by profiling miRNAs from patient serum, plasma, saliva and tissues
prognosis of colon, lung and breast cancers has been linked to specific miRNA profiles.
Circulating miRNAs function as ‘extracellular communication RNAs’ that play an important role in cell proliferation and differentiation - can be used as biomarkers for acute myocardial infarction, congestive heart failure, drug induced liver injury and various malignancies. Resistant to rapid degradation by RNases.
What are the possible applications of ncRNAs as therapeutic agents?
the use of siRNA to selectively silence a defective allele. In fact, there are several ongoing trials using siRNAs to target age-related macular degeneration, acute renal failure and hepatocellular carcinoma.
miRNA manipulation involving either directed silencing or reduction of tumor promoting miRNAs vs. repletion of select tumor-suppressive miRNAs
The silencing or repletion of miRNA to augment chemotherapeutic effect, or the use of global miRNA profiling to classify drug responders and nonresponders in various cancers.
The use of exogenous siRNAs to modulate RNA alternative splicing and correct defective gene expression.
The use of exogenous expression of repressed miRNAs to restore homeostasis.
The use of exogenous small ncRNAs to regulate promoter activity by interfering with epigenetic marks and chromatin formation.
How do ncRNAs function as therapeutic agents?
miRNA therapeutics can either (1) restore function of the miRNA by using synthetic ds miRNAs which harbor chemical modifications to enhance stability and cellular uptake or viral vector based overexpression of miRNAs; (2) Inhibit function of miRNA by using chemically modified antimiR oligonucleotides – transgenic expression of RNA molecule harbouring complementary binding sites to miRNA of interest to block a given miRNA or miRNA family.
Give specific examples of ncRNAs used as therapeutic agents.
MRX34: mimic of tumour supressor miR-34, lost/reduced in most solid tumours/haematological malignancy. Functionsi p53 pathway - inhibits growth by blocking MYC, MET, BCL2 and other oncogenes. Induces cell cycle arrest, apoptosis. Creates a barrier to metastases and abolished chemoresistance
Atu027 - delivers siRNAs to target PKN3 expression. PKN3 involved in malignant cell growth (solid tumours) downstream of chronically activated PI3K pathway
Antisense oligonucleotides (ASOs) against the lncRNA UBE3A antisense transcript (UBE3A-ATS) could represent a feasible therapy for Angelman syndrome. ASOs have been shown to be able to activate expression of the paternal UBE3A allele in cultured mouse neurons and in live mice. Resulted in restoration of UBE3A protein which was sufficient to ameliorate the cognitive defects in mice, and could be a potential treatment for humans.
How are lncRNAs involved in transcriptional factor activation?
Some lncRNAs do not work by direct antisense regulation of their target genes. Gas5 lncRNA works by binding to the DNA-binding domain of the glucocorticoid receptor, thus competing with and modifying the expression of target genes containing genuine glucocorticoid response elements.
How are lncRNAs involved in transcriptional regulation?
Expression of a number of lncRNAs are induced in a p53 dependent manner. These lncRNAs are enriched for the p53-binding motif in their promoters. One of these lncRNAs, lncRNAp21 near the p21 gene, is directly regulated by p53 and subsequently forms a lncRNA-RNP with a nuclear factor to serve as role as a global transcriptional repressor. When p53 induction takes place following DNA damage, this lncRNA represses the expression of downstream genes in the p53 pathway.
How are lncRNAs involved in transcription-mediated silencing?
KA ‘transcriptional interference’, in which the act of transcription of one gene can repress in cis the functional transcription of another gene. The RNA polymerase (RNAPII) initiated from an 'interfering' promoter traverses a 'sensitive' DNA regulatory sequence. E.g. is H19 RNA implicated in imprinting of 11p15 IC1 domain. This lncRNA is transcribed from the paternal chromosome and interferes with transcription of the maternally expressed IGF2 gene.
How are lncRNAs involved in post-transcriptional regulation?
Mediate epigenetic change by recruiting chromatin remodelling complexes
(A) Induction of X inactivation by the Xist lncRNA in female mammals. Xist is expressed from one of the two X chromosomes and induces silencing of the whole chromosome. XIST gene encodes a long ncRNA (17 kb) and recruits the polycomb complex to silence the X chromosome from which it is transcribed. TSIX, another lncRNA, is transcribed from the opposite strand to XIST and regulates XIST levels during X-chromosome inactivation.
(B) HOTAIR. HOTAIR regulates gene expression in trans on a genome-wide scale (including the HOX-D gene cluster) by associating with chromatin-modifying complexes.
(C) Both Air and Kcnq1ot1 are imprinted lncRNAs that are transcribed from the silenced paternal allele, and specifically bind to and recruit the histone H3 lysine 9 methylase G9a in cis to mediate H3K9me3 and transcriptional silencing of Kcnq1 or Igf2r loci, respectively.
How are lncRNAs involved in the modification of alternative splicing?
lncRNA MALAT1 interacts with the SR (serine/arginine)-rich splicing regulatory proteins, which dictate alternative splicing patterns and are regulated by alterations to their
phosphorylation status. The interaction of MALAT1 with these factors results in their relocation to the splicing speckles (the site of mRNA processing) in the nucleus, together with the modification of their phosphorylation state.
How are lncRNAs involved in translational control?
he antisense RNA, BACE1-AS, is transcribed from the opposite strand to BACE1. BACE1AS interacts with the β-site APP (amyloid precursor protein) cleaving enzyme 1 (BACE1) transcript, which is a crucial player in AD (Alzheimer’s disease) pathology. BACE1-AS regulates the expression of BACE1 by increasing BACE1 mRNA stability and generating additional BACE1 through a post-transcriptional feed-forward mechanism.