Structural Basis for Interactions of Long non-coding RNAs and de novo Methyltransferase 3a — ASN Events
  1. Schedule
  2. Session Ten - Poster Session C including Refreshments
  3. Structural Basis for Interactions of Long non-coding RNAs and de novo Methyltransferase 3a

Structural Basis for Interactions of Long non-coding RNAs and de novo Methyltransferase 3a (#342)

James Walshe 1 , Sandro F Ataide 1
  1. University of Sydney, Camperdown, NSW, Australia

The term “junk DNA” was widely use throughout the 1960s to describe the vast noncoding regions within the human genome which scientists of the time believed served little function.   However growing evidence suggest these regions play a vital role in cell regulation.  The recent ENCODE consortium determined that ≈ 80% of the human genome is functionally transcribed and potentially active despite lacking protein-coding capabilities1,2.  Long non-coding RNAs (lncRNA) have been implicated as transcriptional regulators for protein-coding genes through a process that involves the lncRNA directing epigenetic silencing complexes to the targeted loci. One such protein involved in epigenetic silencing is de novo methyltransferase 3A (Dnmt3a).  Dnmt3a has recently been observed to have direct interactions with the antisense lncRNA of the actively transcribed PTEN pseudogene (PTENpg1asRNA).  Suppression of Dnmt3a in 293HEK cells by RNAi results in subsequent activation of the PTEN gene itself suggesting a prominent regulatory role for Dnmt3a and PTENpg1asRNA3.  

Similarly to the PTEN gene, HIV has also been observed to express an antisense transcript (HIVasRNA). Suppression of this antisense transcript results in activation of HIV.   HIVasRNA has been shown to localize with Dnmt3a to the HIV gene promoter4.

The limited knowledge of how Dnmt3a interacts at the molecular level with these lncRNA led us to investigate their molecular mechanism of action.

We are structurally characterizing the interactions between Dnmt3a with PTENpg1 antisense RNA exon 1 and HIV-1 antisense RNA using x-ray crystallography and validating it with via microscale thermophoresis (MST) and EMSA

We have cloned Dnmt3a and several truncations in order to identify the RNA binding domain. We have expressed and are working on purification of each Dnmt3a construct. To date, large-scale overexpression has been best achieved in Escherichia coli Rosetta 2 strain using a 6xHis-tagged MBP truncated Dnmt3a fusion (284-920aa).

Gel shift assays were used to confirm binding of in vitro transcribed PTENpg1asRNA by truncated HisMBP Dnmt3a construct. We have started preliminary crystallization trials to obtain suitable diffracting crystals of the complex.

  1. Bernstein, B. E., E. Birney, et al. (2012). "An integrated encyclopedia of DNA elements in the human genome." Nature 489(7414): 57-74.
  2. Djebali, S., C. A. Davis, et al. (2012). "Landscape of transcription in human cells." Nature 489(7414): 101-108.
  3. Johnsson, P., A. Ackley, et al. (2013). "A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells." Nat Struct Mol Biol 20(4): 440-446.
  4. Saayman, S.,A. Ackley, et al (2014). "An HIV-encoded antisense long non-coding RNA epigenetically regulates viral transcription." Molecular Therapy In Press.