Helge Grosshans

Function and regulation

of animal microRNAs

Introduction
The generation of different cell types and tissues
in an organism, or the responses of cells to envi-
ronmental cues and insults requires faithful yet
dynamic control of gene expression. Numerous
gene regulatory processes acting at both the tran-
scriptional and post-transcriptional levels have
evolved. MicroRNAs (miRNAs) are a class of post-
transcriptional gene regulators that make up >1%
of genes in a typical animal genome. They bind to
target mRNAs through base-pairing and silence
GROUP LEADER
them by mechanisms involving translational
Helge Grosshans
repression and mRNA degradation. As each
helge.grosshans@fmi.ch
miRNA controls a large number of target mRNAs,
miRNAs collectively affect a large fraction of cel-
TECHNICAL / RESEARCH
lular and developmental processes. Consistently,
ASSOCIATES
the levels and activities of miRNAs are themselves
Monika Fasler
highly regulated. Conversely, miRNA dysregula-
Mirela Vitanescu
tion has been implicated in many diseases, most
notably in diverse cancers.
POSTDOCTORAL
We are using cell culture and the roundworm
FELLOWS
Caenorhabditis elegans to study how miRNAs are
Nicolas Antih
made and regulated. In addition to exploiting the
Ingo Bssing*
traditional strengths of C. elegans in genetics and
Saibal Chatterjee*
cell biology, we are developing and applying bio-
Manuel De la Mata
chemical assays. These complementary approaches
Takashi Miki
allow us to obtain insights into both the molecu-
lar mechanisms of new pathways and their physi-
PhD STUDENTS
ological relevance. Thus, we have been able to
Florian Aeschimann
identify an miRNA degradation pathway that pre-
Hrishikesh Bartake
vents detrimental increases in mature miRNA
Matyas Ecsedi
levels. We have also shown that mRNAs are not
Gert-Jan Hendriks
simply inert targets of miRNAs but that miRNAs
Benjamin Hurschler*
and mRNAs form a network of mutual regulation.
Magdalene Rausch
We are now exploring the mechanistics of miRNA
Hannes Richter
degradation and its roles in development and dis-
Stefan Regger
ease. We also continue to study the issue of how
miRNAs silence their targets mechanistically in
UNDERGRADUATE
vivo and whether this process is regulated.
Melanie Hunkeler*

*left the group

58 FMI Report 2011/2012

REGULATION REVERSED: TARGET mRNAS
MODULATE miRNA LEVELS
S.Chatterjee, M.Fasler, I.Bssing, N.Antih,
M.delaMata, M.Vitanescu
Biogenesis of miRNAs involves their transcription
as long capped and polyadenylated primary
miRNAs and processing by the nucleases Drosha
and Dicer. The resulting miR:miR* duplex is sub-
sequently disassembled and the miRNA guide
strand, termed miR, loaded into a protein of the
AGO family, constituting the core of the repressive
miRNA-induced silencing complex miRISC. The
miR* passenger strand is discarded. Many or all of
these steps can be regulated and maintain faithful
miRNA expression levels.
Recently, we identified XRN-2 as an important
factor for miRNA homeostasis in C. elegans. How-
ever, rather than affecting miRNA biogenesis, this
exoribonuclease (RNase) mediates degradation of
mature miRNAs. When we recapitulated miRNA
turnover in vitro, we found that single-stranded but
not partially double-stranded miRNAs were de-
graded. In particular, the miR:miR* duplex that
results from processing of the pre-miRNA by Dicer
and mature miRNA in a complex with target RNA
were resistant to degradation.
To determine whether this mechanism is physi-
ologically relevant, we modulated miRNA target
levels in vivo by over-expressing artificial targets or
depleting endogenous targets. As predicted from the
in vitro data, this resulted in corresponding changes
in miRNA levels, which increased or decreased,
respectively. Similarly, sequence-related miRNAs
competed with each other for targets such that over-
expression of one family member reduced the levels
of another. Finally, the presence of a target of miR-
241* elevated its levels, although it was previously
assumed that miRNA strand selection is determined
largely by sequence and structure features and thus
is invariant for a given miR:miR* duplex.
Given these results, we propose that the combi-
nation of mature miRNA degradation and its pre-
vention by Target-Mediated MiRNA Protection
(TMMP) provide a mechanism that (i) proof-reads
AGO-loaded miRNA for function, (ii) allows dy-
namic miRNA expression, and (iii) possibly com-
prises a mechanism of miRNA evolution. Non-
functional RNAs that are loaded into AGO (e.g.,
those derived from spurious transcription of hair-
pin structures in the genome) would thus be elimi-
nated from AGO and prevent its 'clogging-up';
upon acquisition of targets they would be function-
ally selected and stabilized. Ongoing research is
aimed at determining how target architecture and
expression levels determine TMMP capacity. Using
cell culture, we are further exploring how miRNA
turnover occurs in mammalian cells and what effect
targets have on mammalian miRNAs.
XRN-2: CHARACTERIZATION OF A miRNA-
DEGRADING ENZYME
S.Regger, H.Richter, T.Miki
To understand the molecular mechanism and de-
velopmental function of miRNA turnover, we are
employing diverse approaches to characterize in
more detail the XRN-2 RNase and its effect on
miRNA levels. In addition to employing co-immu-
noprecipitation and related techniques to reveal
physical interaction partners of XRN-2, we have
generated a temperature-sensitive (ts) xrn-2 mu-
tant (Fig.1) to identify genetic interaction part-
ners. By running modifier screens at permissive
and restrictive temperatures, we will identify both
enhancers and antagonists of XRN-2 activity, as
well as factors acting in parallel miRNA turn-over
pathways.
To study the biochemistry of XRN-2 in more
detail, we are using bacterially expressed recom-
binant XRN-2, a 110-kDa protein. We are com-
Incubation time at restrictive temperature
3h 5h 7h
wild-type
xrn-2(ts)
bining in vitro and in vivo mutational dissection Fig.1. XRN-2 is required
of XRN-2 to identify relevant protein domains. for embryonic develop-
Finally, we are using targeted and genomics ap- ment. Two-cell stage em-
bryos were dissected from
proaches to define the substrate spectrum of
mothers grown at permis-
XRN-2. Our long-term goal is to reconstitute sive temperature and
miRNA release from AGO and subsequent miRNA incubated for the indicated
turnover in vitro, and to link individual miRNA times at the restrictive
turnover events to developmental processes or temperature. While wild-
physiological responses to environmental cues. type embryogenesis is
largely complete within 7h,
xrn-2(ts) mutants arrest
IDENTIFICATION OF NOVEL miRNA
early
PATHWAY GENES
M.Ecsedi, M.Rausch, F.Aeschimann,
G.-J.Hendriks
During the past 10 years, we have witnessed an
explosion in knowledge of miRNAs that has
yielded a well-supported model of the core steps
of miRNA biogenesis and function. However,
many details of these processes are still lacking, as
well as insights into the events regulating them.
To fill these gaps, we are using genetic interaction
screens to identify factors that modulate miRNA
activity. For instance, a mutation in let-7 impairs
FMI Report 2011/2012 59
Epigenetics
A hypodermal promoter
GFP
let-7 target 3UTR
wild-type
let-7(n2853)
B
let-7(n2853);
mock(RNAi)
let-7(n2853);
xrn-2(RNAi)
GFP DIC
Fig.2. Live-imaging of the function of this miRNA and causes C. elegans
miRNA activity. A. 3 UTR to die by bursting. After successful completion of
targeted by the let-7 a pilot experiment, where we knocked down
miRNA silences an actively
>2,000 genes located on C. elegans chromosome I
transcribed GFP reporter in
the skin (hypodermis).
to identify those that restored the viability of let-7
Repression is lost in let- mutant animals, we have now expanded this
7(n2853) mutant animals. screen to a genome-wide level. Through examina-
B. Depletion of negative tion of >16,000 genes, we found approximately
regulators of let-7 such 200 suppressors of let-7 lethality. To classify the
as xrn-2 restores reporter suppressors further, we ran them through second-
gene silencing
ary screens that scored restoration of a) let-7-de-
pendent skin-cell differentiation, and b) let-7-
mediated mRNA silencing (Fig. 2). A large fraction
of suppressor genes scored positive in these
secondary screens. We are currently focusing our
efforts on understanding the functions of the
ca. 30 candidates that scored positive in both
assays.
Selected publications
1. Hurschler BA, Harris DT, Grosshans H 4. Chatterjee S, Grosshans H
(2011) The type II poly(A)-binding (2009) Active turnover modulates ma-
protein PABP-2 genetically interacts ture microRNA activity in C.elegans.
with the let-7 miRNA and elicits hetero- Nature 461:546-549
chronic phenotypes in Caenorhabditis
elegans. Nucleic Acids Res 5. Ding XC, Grosshans H (2009)
39:5647-5657 Repression of C. elegans microRNA
targets at the initiation level of
2. Chatterjee S, Fasler M, Bssing I, translation requires GW182 proteins.
Grosshans H (2011) Target-mediated EMBO J 28:213-222
protection of endogenous microRNAs
in C. elegans. Dev Cell 20:388-396
3. Bssing I, Yang J-S, Lai EC,
Grosshans H (2010) The nuclear export
receptor XPO-1 supports primary
miRNA processing in C. elegans and
Drosophila. EMBO J 29:1830-1839
60 FMI Report 2011/2012
We expect the let-7 suppressor screen to yield
insights into both regulation and mode of action
of miRNAs. To address the latter issue more di-
rectly, we are complementing this screen with an
investigation of interaction partners of the C. ele-
gans GW182 proteins AIN-1 and AIN-2, which
have crucial activities in mediating miRNA-in-
duced mRNA degradation and translational re-
pression.
DEVELOPMENTAL FUNCTION OF THE let-7
miRNA
B.A.Hurschler, H.Bartake, M.Ecsedi and
M.Rausch, in collaboration with D.T.Harris
(HHMI/MIT, Cambridge, USA)
In C. elegans, the let-7 miRNA represses cell pro-
liferation and promotes stem cell differentiation,
thus controlling temporal patterning during the
transition from late larval to adult stages. Mam-
malian let-7, which is identical in sequence to C.
elegans let-7, shares these functions and, thus, acts
as a tumor suppressor. How let-7 exerts its activi-
ties is only partially understood.
We recently identified pabp-2, the C. elegans or-
tholog of type II poly(A)-binding protein PABP2/
PABPN1, as a genetic interaction partner of let-7.
In mammals and flies, PABP2 is thought to func-
tion in general mRNA metabolism by promoting
poly(A) tail formation. Nonetheless, we found
that PABP-2 levels were developmentally regu-
lated in a let-7-dependent manner, although pre-
sumably indirectly. Moreover, depletion of C.
elegans PABP-2 by >80% suppressed loss of let-7
activity and, in let-7 wild-type animals, led to pre-
cocious differentiation of seam cells. In contrast,
this level of PABP-2 depletion did not significantly
impair larval viability, mRNA levels or global
translation. Thus, unexpectedly the bulk of
PABP-2 appears to be dispensable for general
mRNA metabolism in the larva and may instead
have more restricted developmental functions.
This may also help to explain why the phenotypes
associated with the human PABP2 mutation in
OPMD (oculopharyngeal muscular dystrophy)
selectively affect muscle cells.
Using the results from the genome-wide let-
7(n2853) screen described above and candidate
gene testing, we are now focusing on understand-
ing how let-7 coordinates regulation of cell dif-
ferentiation and proliferation. p