Enzymes with alter egos

By Graeme O'Neill
Wednesday, 12 February, 2014

Enzymes with alter egos

Enzymes are well known for their housekeeping role in cells, but some enzymes bind RNA and might play a significant role in linking intermediary metabolism to gene expression via post-transcriptional regulation. 

Professor Matthias Hentze, of the European Molecular Biology Laboratory (AMBL) in Heidelberg, Germany, and Professor Thomas Preiss, now Professor of RNA Biology at the Australian National University’s (ANU’s) John Curtin School of Medical Research, will be taking their longstanding research collaboration to a new level, after years of communicating via Skype.

After giving his plenary lecture to the 2013 Genome meeting, Hentze, who is bringing five of his top people with him to Lorne, will head to the surf and sand of Kioloa for a working retreat with his team and members of Preiss’ team. Preiss was a postdoctoral researcher in Hentze’s laboratory from 1995 to 2002.

Binding RNA

Their main topic for discussion will be enzymes with alter egos - recent decades have seen sporadic reports that certain enzymes, as well as mediating classic metabolic reactions in cells, act as RNA-binding proteins.

“We work together in two overlapping areas,” Preiss said. “One is the question of the regulatory connection between gene expression and cellular metabolism - we see that as one of the big challenges, because it requires us to combine the largely separate disciplines of molecular biology and biochemistry.

“Over the past few decades, a number of research groups have made the curious observation that certain metabolic enzymes have an RNA-binding function, but in most cases the physiological role of this function has remained unclear.”

Post-transcriptional regulatory networks

Preiss says that in 1987 Hentze co-discovered iron-responsive elements (IREs), the first regulatory elements found in mammalian messenger RNAs (mRNAs). They occur in the untranslated regions (UTRs) of mRNAs encoding for proteins involved in iron transport and storage and interact with iron regulatory proteins (IRPs).

Remarkably, one of the IRPs, IRP1, is a bifunctional protein that in iron-replete cells ligates an iron-sulfur cluster to function as an aconitase enzyme, catalysing the stereo-specific isomerisation of citrate to isocitrate. Its alter ego in iron-deficient cells adopts its RNA-binding (apo-enzyme) conformation.

Preiss says that while enzymes of course have been widely studied for their regular, ‘housekeeping’ roles in cell metabolism, little attention has been paid to their RNA-binding functions.

In an opinion article in Trends in Biochemistry in 2010, Hentze and Preiss suggested that RNA-binding enzymes might frequently be players in a class of post-transcriptional regulatory networks, linking intermediary metabolism to gene expression, through interactions between RNA, enzymes and metabolites (REMs).

mRNA interactome capture

Further, they challenged the view that enzymes are mainly post-translationally regulated by direct feedback from fluctuating levels of their own metabolites, changes in nutrient availability, redox state, oxygen tension or stress.

“One of the things we would like to do is to identify more broadly which enzymes in different cells can bind mRNA,” Preiss said.

“Matthias came up with a clever way to identify all cellular RNA-binding proteins, that he calls ‘mRNA interactome capture’.

“It uses ultraviolet light to cross-link RNA-binding proteins their RNA targets in vivo. Oligo dT beads are then used to capture mRNAs by their polyA tails, allowing the attached proteins to be recovered and identified by mass spectrometry.”

Preiss says the technique, published in a recent paper in Cell, has revealed a previously unsuspected abundance of mRNA-binding proteins in eukaryotic cells, pointing to the existence of a system of RNA ‘operons’ that post-transcriptionally regulate the activity of messenger RNAs with related functions after they are released into the cytoplasm.

“Conceptually, mRNA-binding protein functions are quite similar to the roles shown for microRNAs,” Preiss said. “By coordinating the functions of messenger RNAs, RNA-binding proteins endow cells with extra regulatory potential.”

A new window on genetic disease

More than 1100 proteins in cultured human and other eukaryotic cells have now been identified as having RNA-binding activity - at least 350 of them were not previously known to have RNA-binding roles.

Preiss says the collaboration between the ANU and EMBL teams is filling out details of the interactions within these post-translational regulatory networks by investigating, in specific cellular contexts, what groups of mRNAs interact with RNA-binding proteins - “RNA-binding proteins operate in parallel, and interact cooperatively”, he said.

In a recent review in Trends in Genetics, Preiss and Hentze and two other colleagues, Alfredo Castello and Bernd Fischer, further described how errors in these networks may explain a range of inherited disorders.

Complex systems are fraught with potential to malfunction, and the ANU-EMBL team’s growing catalogue of RNA-binding proteins has opened up a new window on genetic disease.

The ANU-EMBL paper notes that the mRNA interactome contains many proteins that, in mutant form, are associated with Mendelian disorders - predominantly of neurological and sensory systems, muscular atrophies, metabolic disorders and cancers.

The specific mutations involved in such disorders are scattered through the domain architectures of the proteins. Many occur in non-classical RNA-binding domains and in unfolded epitopes.

In some cases, the mutations might perturb previously unrecognised RNA-related functions of the proteins.

“We also expect that mRNA interactome capture approaches will aid further exploration of RNA systems biology in varied physiological and pathophysiological settings,” the authors write.

Post-transcriptional regulation hubs

A wealth of literature now attests to the role of RNA-binding proteins, as well as noncoding RNAs including microRNAs, in directing and regulating the post-transcriptional fate of messenger RNAs in the nucleus and cytoplasm.

RNA binding proteins variously influence splicing and the formation of the 3’ regions of mRNAs, as well as post-transcriptional editing of mRNAs, their localisation in cell compartments, translation into protein and mRNA turnover, “often in a dynamic and cell type-specific manner”.

The authors say the recent discovery of widespread, regulated, alternative mRNA 3’-end formation in many cellular and disease contexts underscores the importance of 3’UTRs as hubs of post-transcriptional regulation - and, by implication, as likely foci for mutation-induced mischief.


Lorne conference line-up

Here’s the line-up for the Lorne conferences for 2014, to be held at Mantra Lorne on the Victorian south coast.
19th Lorne Proteomics Symposium
February 6-9
39th Lorne Conference on Protein Structure and Function
February 9-13
26th Lorne Cancer Conference
February 13-15
35th Lorne Genome Conference
February 16-19
Lorne Infection and Immunity


Image: Professor Thomas Preiss (L) is Professor of RNA Biology at the Australian National University's John Curtin School of Medical Research and Professor Matthias Hentze (R) is co-director and co-founder of the Molecular Medicine Partnership Unit between the European Molecular Biology Laboratory (EMBL) and the Medical Faculty of Heidelberg University. After completing a PhD at the University of Newcastle Upon Tyne, Preiss spent seven years as a postdoctoral researcher in Professor Hentze's EMBL laboratory, before being appointed laboratory head at the Victor Chang Cardiac Research Institute at Sydney. After completing an MD at the University of Munster in North Rhine-Westphalia, Hentze undertook a postdoctoral appointment at the National Institutes of Health and then joined EMBL Heidelberg as a Group Leader in 1989. He has been a senior scientist with EMBL since 1998, specialising in metabolism, RNA biology and molecular medicine.

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