1. “Gene Expression Regulation”
Lesson 7

2. LONG NON CODING RNAs

3. LONG NON CODING RNAs (LncRNAs)
By definition: RNA that are more than 200 nucletides long
LncRNAs evolved more than protein coding RNAs (mRNAs).
While small ncRNAs are very conserved in different species, LncRNAs are not very conserved.
Different selection: the regions of LncRNAs that are well conserved either have a particular structure or are engaged in sequence-specific interactions with other nucleic acids.

5. LncRNAs play a role in several biological processes:
Chromatin modifications: Xist, HOTAIR
Interaction with RNApolII transcription
Post transcriptional modifications: for example they modulate mRNA maturation
They can be processed and therefore originate small ncRNAs that can either trigger the degradation or the impairment of translation of their targets.

6. Long non-coding RNAs (lncRNAs) are a large and diverse class of transcribed RNA molecules with a length of more than 200 nucleotides that do not encode proteins (or lack > 100 amino acid open reading frame). lncRNAs are thought to encompass nearly 30,000 different transcripts in humans, hence lncRNA transcripts account for the major part of the non-coding transcriptome. lncRNA discovery is still at a preliminary stage. There are many specialized lncRNA databases, which are organized and centralized through RNAcentral.

7. lncRNAs can be transcribed as whole or partial natural antisense transcripts (NAT) to coding genes, or located between genes or within introns. Some lncRNAs originate from pseudogenes.
lncRNAs may be classified into different subtypes (Antisense, Intergenic, Overlapping, Intronic, Bidirectional, and Processed) according to the position and direction of transcription in relation to other genes.

8. LncRNA expression is developmentally regulated, can be tissue- and cell-type specific, and can vary spatially, temporally, or in response to stimuli.
In general, the expression level of lncRNA is at least one order of magnitude below that of mRNA. Many lncRNAs are located exclusively in the nucleus, but some are cytoplasmic or are located both in the nucleus and in the cytoplasm.

10. LncRNAs are key regulators of embryonic pluripotency and differentiation during development, but may modulate also process in adult life.
LncRNAs may contribute to nuclear organization, epigenetic regulation, post-transciptional regulation and mRNA splicing.

13. Modulation by transcription itself
Modulation as antisense
Modulation via protein complex

19. HOTAIR
HOX Antisense Intergenic RNA (HOTAIR) is a lncRNA of 2.3 Kb; it is an antisense RNA transcribed from the Homebox C (HOXC) cluster on chromosome12 and it is coexpressed along with HOXC genes. HOTAIR acts in trans on chromosome 2 repressing the transcription of HOXD locus.
HOTAIR interacts at the same time with PRC2 (that allows H3K27 methylation) and with the specific lysine demethylase 1 complex (LSD1, demethylation of H3K4).

20. Gibb et al. Molecular Cancer 2011 10:38 doi:10.1186/1476-4598-10-38

21. Proposed mechanism of HOTAIR mediated gene silencing of 40 kb of the HOXD locus, which is involved in developmental patterning. The HOTAIR lncRNA is transcribed from the HOXC locus and functions in the binding and recruitment and binding of the PRC2 and LSD1 complex to the HOXD locus. For clarity, only the PRC2 complex is indicated in the above figure. Through an undetermined mechanism, the HOTAIR-PRC2-LSD1 complex is redirected to the HOXD locus on chromosome 2 where genes involved in metastasis suppression are silenced through H3K27 methylation and H3K4 demethylation. This drives breast cancer cells to develop gene expression patterns that more closely resemble embryonic fibroblasts than epithelial cells.
Gibb et al. Molecular Cancer :38 doi: /

22. ANRIL
ANRIL is a lncRNA transcribed by the chromosome region 9p21 (an hotspot locus associated to several pathologies).
ANRIL modulates the expression of the in cis oncosuppressor genes CDKN2A e B.
It is transcribed by RNA Pol II, and has several splicing products. It mainly acts at the epigenetic level by interacting with the Polycomb repressive complex (PRC1 and PRC2).
ANRIL acts in cis on CDKN2A e B genes.
It binds PRC1 e PRC2 that promote H3K27 methylation on CDKN2A and 2B locus triggering epigenetic silencing of gene expression.

27. GAS5
The lncRNA GAS5, Growth arrest-specific transcript 5, has been originally identified as a potential tumor suppressor gene as its level of expression in extremely low in actively proliferating cells, while it is high in those that are arrested in response to of nutrients or growth factors deprivation.
It acts as a decoy: it interacts with the DNA binding domain of Glucocorticoid receptor (GR) activated by the ligand binding. GAS5 has a “GRE-mimic” sequence and may therefore sequester GR by competing for its binding to the GRE sequences located on the promoters of GR target genes (including the antiapoptotic genes cIAP2, SGK1).
In this way GAS5 represses the signal mediated by glucocorticoids,

28. Figure 13: Interaction model of Gas5 RNA “GRE” with GR DBD and the effect of Gas5 on GRa-induced transcriptional activity.
A: 3-Dimenstional structure of Gas5 “GRE”-mimic and its interaction model with the GR DBD. From (252).
B: Schematic model of the effect of Gas5 on GR-induced transcriptional activity.
Gas5 accumulated in response to growth arrest/starvation binds GR DBD and attenuates GR-induced transcriptional activity by competing with DNA GREs located in the promoter region of glucocorticoid-responsive genes.

30. MALAT1
The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a bona fide long noncoding RNA (lncRNA). MALAT1, also known as nuclear-enriched transcript 2 (NEAT2), was discovered as a prognostic marker for lung cancer metastasis but also has been linked to several other human tumor entities. MALAT1 is an interesting target for antimetastatic therapy in non-small cell lung carcinoma.
Two alternative modes of action have been proposed for MALAT1: 1) regulation of gene expression or 2) regulation of alternative splicing.

38. I Signaling
The transcription of certain LncRNAs is very temporal and tissue specific and may selectively occur in response to specific stimuli.
Thus LncRNAs can serve as molecular signals and can act as markers of functionally relevant biological events.
II Decoys
The molecular type decoy activity takes place when specific LncRNAs are transcribed and then bind to and titrate away protein factors.
Decoy LncRNAs can “sponge” protein factors such as transcription factor and chromatin modifiers. This leads to broad changes in the cell’s transcriptome.

39. III Guides
LncRNAs can be molecular guides by localizing particular ribonucleo proteins to specific chromatin targets.This activity can change gene epression either in cis (on neighboring genes) or in trans (on distant genes) that cannot be predicted by just the lncRNA sequence itself.
IV Scaffolds
Assembly of complex protein complexes can be supported by LncRNAs, linking factors to together form new functions. Some lncRNAs have different domains that bind distinct protein factors that all together, may impact on transcriptional activation o repression.

41. LncRNAs participate to a widevariety of biological processes
Orly Wapinski and Howard Y. Chang, Long noncoding RNAs and human disease. Trends Cell Biol Jun;21(6):

46. You want to demonstrate that p53 is a tumor suppressor gene
You want to demonstrate that p53 is a tumor suppressor gene. Which in vitro experiment can you do? How does it work?