Cellular functions of the microprocessor complex
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Cordiner2016.docx (8.809Mb)
Date
29/11/2016Author
Cordiner, Ross Andrew Alex
Metadata
Abstract
DGCR8 (DiGeorge critical region 8) protein constitutes part of the
Microprocessor complex together with Drosha, and is involved in the nuclear phase of
microRNA (miRNA) biogenesis. DGCR8 recognises the hairpin RNA substrates of
precursor miRNAs through two double-stranded RNA (dsRNA) binding motifs and acts
as a molecular anchor to direct Drosha cleavage at the base of the pri-miRNA hairpin.
Recent characterisation of the RNA targets of the Microprocessor by HITSCLIP
of DGCR8 protein revealed that this complex also binds and regulates the stability
of several types of transcripts, including mRNAs, lncRNAs and retrotransposons. Of
particular interest is the binding of DGCR8 to mature small nucleolar RNA (snoRNA)
transcripts, since the stability of these transcripts is dependent on DGCR8, but
independent of Drosha. This raises the interesting possibility that there could be
alternative DGCR8 complex/es using different nucleases to process a variety of cellular
RNAs.
We performed mass spectrometry experiments and revealed that DGCR8 copurifies
with subunits of the nucleolar exosome, which contains the exonuclease RRP6.
We demonstrated DGCR8 and the exosome form a nucleolar complex, which degrade the
mature snoRNAs tested within this study. Interestingly, we also show that
DGCR8/exosome complex controls the stability of the human telomerase RNA
component (hTR/TERC), and absence of DGCR8 creates a concomitant telomere
phenotype.
In order to identify the RNA targets of the DGCR8/Exosome complex on a
global scale we performed iCLIP of endogenous and overexpressed RRP6 (wild-type and
a catalytically inactive form). Thus, intersection of CLIP datasets from DGCR8 and RRP6
identified common substrates; accordingly snoRNAs were the most represented. In
addition, we identified the cellular RNA targets of the RRP6 associated human exosome.
The use of a catalytically inactive form of RRP6 stabilised important in vivo interactions
that are highly dynamic and transient and also highlighted the role of RRP6-mediated
trimming of 3’flanks of immature non-coding RNAs. We will present a global view of the
RNA-binding capacity of the RRP6-associated exosome.
In sum, we identified a novel function for DGCR8, acting as an adaptor to
recruit the exosome to structured RNAs and induce their degradation. Moreover, we have
identified DGCR8-depenedent substrates of the exosome and have demonstrated the
requirement of RRP6 for 3’ processing of ncRNAs.