Modulation of Ago-miRNA regulatory networks by cis-sequence elements and target competition

278 Pages·2014·31.14 MB·English

Modulation of Ago-miRNA regulatory networks by cis-
sequence elements and target competition
ARCIVES
by
SACHUSET79 INWT1UtE
Andrew D. Bosson OF TECHNOLOGY
MAY 2 5 2014
B.S. Genetics, University of Georgia (2008)
LIBRARIES
Submitted to the Department of Biology
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
June 2014
C 2014 Massachusetts Institute of Technology
All rights reserved
Signature redacted
Signature of Author:
Andrew D. Bosson
Department of Biology
May 23, 2014
redacted
Signature
Certified by:
/ 1 1
Phillip A. Sharp
Institute Professor of Biology
Signature redacted Thesis supervisor
Accepted by:
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Michael T. Hemann
Associate Professor of Biology
Chair, Biology Graduate Committee
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Modulation of Ago-miRNA regulatory networks by cis-sequence elements and
target competition
by Andrew D. Bosson
Submitted to the Department of Biology on May 23, 2014 in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy in Biology
ABSTRACT
microRNAs (miRNAs) are short non-coding RNA molecules that function as post-
transcriptional regulators of gene expression in a wide range of organisms and biological
processes. Each miRNA guides Argonaute (Ago) protein complexes to target and repress
hundreds of genes in a sequence-dependent manner. To identify all targets of miRNA
regulation, we performed UV crosslinking and immunoprecipitation (CLIP) of Ago complexes in
mouse embryonic (ESC) and mesenchymal (MSC) stem cell lines. We also captured the
genome-wide miRNA-independent binding footprint of Ago by performing CLIP in cells that
lack Dicer, an enzyme required for mature miRNA biogenesis. We surprisingly found that Ago
bound a similar set of genes in the absence of Dicer, and this overlap in target genes was due
partially to residual, unprocessed miRNAs in the Dicer KO cells. Other potential sites of
miRNA-independent Ago interactions, such as histone transcripts and poly-A cleavage and
polyadenylation sites, were also identified.
One Ago CLIP dataset revealed the enrichment for a G-rich sequence motif at Ago target
sites. We later demonstrated that the G-motif is not directly bound to Ago but rather is enriched
near miRNA-guided Ago binding sites. Its presence near miRNA target sites is associated with
stronger repression of Ago-miRNA targets. Fortuitously, the original Ago CLIP dataset that
identified the G-motif was later shown to likely contain target sites of another co-
immunoprecipitating RNA binding protein (RBP). Using mass spectroscopy of Ago antibody
immunoprecipitations from Ago KO cells, we identified a list of interacting RBPs that could
potentially augment Ago-miRNA activity through the G-motif.
To investigate target competition in miRNA networks, we related our CLIP analysis of
genome-wide, quantitative Ago binding to measurements of absolute miRNA and target RNA
concentrations. We found that all miRNAs other than the miR-290 family in ESCs and let-7
family in MSCs were expressed at concentrations below their total target pool. However, 8-12
miRNA families were expressed at near or greater than equimolar ratios with their pool of high
affinity targets, and this affinity-partitioned stoichiometry led to significant Ago accumulation
and repression of high affinity target sites despite little consequential binding at low affinity
sites. Single-cell reporter assays demonstrated that high expressed miRNAs are not susceptible to
physiological inductions of competing target transcripts but targets of lower expressed miRNAs
are derepressed in a weakly threshold-like manner upon increased target pool levels.
In summary, we identify a network of confidently bound targets of miRNA regulation in
ESCs and MSCs, reveal the extent of miRNA-independent binding in these two cell types,
provide a list of potential miRNA enhancer RBPs, and create a quantitative context for
evaluating target competition in miRNA networks.
Thesis supervisor:
Phillip A. Sharp, Institute Professor of Biology
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Table of Contents
C hapter 1. Introduction...................................................................................................... 7
miRNAs: discovery, biogenesis, and mechanisms of action ........................................... 8
m iRN A s: biological function........................................................................................ 10
In stress ....................................................................................................................... 12
In developm ental canalization. ................................................................................ 14
m iRN A s: regulation of activity...................................................................................... 15
Factorsa ffecting m iRNA activity at individual sites ............................................... 15
Factors affecting m iRNA activity globally. ............................................................... 16
RBP-bound cis regulatory elements......................................................................... 18
Target competition.................................................................................................... 21
References......................................................................................................................... 25
Chapter 2. Genome-wide identification of Ago2 binding sites from mouse embryonic
stem cells w ith and w ithout m ature m icroR NA s ............................................................... 39
A bstract ........................................................................................................................... 40
Introduction ..................................................................................................................... 41
Results ............................................................................................................................. 42
D iscussion ....................................................................................................................... 55
M aterials and M ethods ................................................................................................. 60
References ....................................................................................................................... 62
Figures ............................................................................................................................. 68
Chapter 3. miRNA-dependent and -independent binding of Ago2 to mRNA targets.... 81
A bstract ........................................................................................................................... 82
Introduction ..................................................................................................................... 83
Results ............................................................................................................................. 85
D iscussion ..................................................................................................................... 100
M aterials and M ethods .................................................................................................. 103
References ..................................................................................................................... 107
Figures ........................................................................................................................... 112
Chapter 4. Quantitation of endogenous miRNA and target pool concentrations predicts
ceRN A effects based on affinity com petition.................................................................... 147
A bstract ......................................................................................................................... 148
Introduction ................................................................................................................... 149
Results ......................................................................................... 152
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D iscussion ..................................................................................................................... 165
Materials and Methods ............................................ 171
References ..................................................................................................................... 177
Figures ........................................................................................................................... 184
Tables of miRNA and target pool concentrations in ESC and MSC ............................ 208
Conclusions and Future D irections ................................................................................... 209
Appendix. Supplementary Information related to chapter 2................................................ 215
Biographical N ote................................................................................................................ 276
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Acknowledgements
This work would never have been completed without the generous support, both scientific and
otherwise, of all the members of the Sharp lab. A helping hand was always extended to me when
in need of reagents, protocols, and general advice.
To Phil, for the faith he placed in me to act as an independent scientist, for the example of
quantitatively thinking about biological processes, for the general openness to enter his office
and discuss whatever is on our minds, for creating a great environment to learn and work, and for
the amazing opportunity to be a part of paradigm shifting research.
To the members of Room 411, for making coming into lab a pleasurable and fruitful experience
from day one. To Arvind, for our meandering discussions of interesting science and crazy
experiments that shaped my creative view of research. To Sara, for always being excited about
storytime and keeping things interesting in lab. To Mohini, for your calming influence on the
room and willingness to always stop and discuss ideas even when you are "in the zone." To
Albert, for the inspiration to push through difficult times in research and follow your dreams of
tackling the difficult questions in science.
To my collaborator Jesse, who taught me how to frame a research paper and analyze complex
datasets, while putting up with my crazy schedule.
To my committee members, Professors David Bartel, Laurie Boyer, and Chris Burge, for sage
advice offered in meetings and pushing me to think critically and focus on the major themes of
my research.
Finally, to my family, who have given me so much to be thankful for. I love them all deeply and
could not have gotten to where I am today without them. Particularly to my mother, who was
always excited to talk with me about my research and even take the time do independent study to
learn about what it is I do every day in lab.
-Andrew Bosson
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To my Grandparents,
Jim and Betty Colmey
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Chapter
1
Introduction
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miRNAs: discovery, biogenesis, and mechanisms of action
As with most exciting discoveries in science, the story of miRNAs began with a surprise.
Victor Ambros's group cloned the lin-4 gene in C. elegans and demonstrated that it was critical
for faithful developmental timing in worms. However, lin-4 did not encode a protein; rather, it
produced a non-coding RNA that base paired to the 3'UTR of the developmentally essential
gene, lin-14 (Lee et al., 1993; Wightman et al., 1993). This complementary base pairing resulted
in down-regulation of Lin- 14 protein at a precise stage in development.
Since that discovery, hundreds of similar short non-coding RNAs, termed miRNAs, have
been identified in over 200 species including worms, flies, mammals, plants, fish, algae, and
viruses (Kozomara and Griffiths-Jones, 2014; Lagos-Quintana et al., 2001; Lau, 2001; Lee and
Ambros, 2001; Pasquinelli et al., 2000; Reinhart et al., 2000). Technology allowing deep
sequencing of smallRNAs quickly increased the number of annotated mammalian miRNAs from
145 in the year 2004 (Bartel, 2004) to 390 in 2006 (Mineno et al., 2006) to over 500 miRNAs
identified across diverse mouse tissues in 2010 (Chiang et al., 2010). In a manner similar to that
seen for some of the proposed lin-4:lin-14 interactions, these miRNAs are collectively predicted
to target over 60% of protein-coding genes in the mammalian genome through complementary
base pairing of the miRNA to conserved sequences in 3'UTRs (Friedman et al., 2009). Perfectly
complementary pairing with a target results in enzymatic cleavage of the target transcript (Yekta
et al., 2004). However, the vast majority of miRNA interactions involve imperfect, or bulged,
pairing that results in translational inhibition and enhanced decay of the target (discussed below).
miRNAs are initially generated as longer, hairpin-forming precursors, called pri-miRNAs
(Bartel, 2004; Carthew and Sontheimer, 2009), most often by Pol 1I transcription (Lee et al.,
2004). About 40% of pri-miRNA genes are located in introns of protein-coding genes, but most
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are independent transcriptional units (Rodriguez et al., 2004). Multiple miRNAs can be encoded
as clusters in one pri-miRNA transcript (Baskerville and Bartel, 2005; Lau, 2001). In mammals,
pri-miRNAs are furthered processed into -60-70nt pre-miRNAs by a large protein complex,
composed principally of the Drosha endoribonuclease and its double stranded RNA binding
protein partner DGCR8 (Han et al., 2004; Lee et al., 2003). The pre-miRNA is transported from
the nucleus into the cytoplasm by Exportin-5 (Lund et al., 2004) where it is recognized by Dicer.
Dicer is an RNase III related enzyme that cleaves the pre-miRNA hairpin in two steps to produce
5p-arm and 3p-arm miRNA strands, each -22 nucleotides (nt) in length (Bernstein et al., 2001;
Gurtan et al., 2012; Hutvitgner et al., 2001; MacRae et al., 2007). One of the two resulting
mature miRNA strands is transferred from Dicer to an Argonaute protein (Ago, described below)
through the interaction of another RNA binding protein, TRBP (Wang et al., 2009). The
decision of whether the 5p or 3p arm miRNA becomes loaded into Ago is primarily determined
by which arm has weaker thermodynamic stability at the 5' end (Khvorova et al., 2003; Schwarz
et al., 2003).
miRNAs are loaded into Ago proteins, key substituents of the RNA induced silencing
complex (RISC) (Hutvaigner and Zamore, 2002; Liu et al., 2004; Meister et al., 2004). There are
four Ago proteins in mammals (Ago 1-4), and they are mostly redundant, such that one of the
four Ago proteins is both necessary and sufficient for miRNA-mediated repression (Hutvagner
and Simard, 2008; Pillai, 2004; Su et al., 2009). Structurally, Ago preforms the miRNA strand
into a helix that exposes positions 2-6 for base pairing with RNA target transcripts (Parker et al.,
2005; Wang et al., 2008b). This conformation results in Ago-miRNA complexes binding to and
repressing mRNA transcripts based predominantly on pairing of positions 2-7 of the miRNA,
known as the "seed" (Brennecke et al., 2005; Doench, 2004; Kloosterman et al., 2004; Lai et al.,
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2005; Lewis et al., 2003). The seed sequence is used to group miRNAs into families, which
share identical seed sequences and therefore target similar sets of genes (Grimson et al., 2007).
Once bound to an mRNA target, Ago acts with the GW- 182 family of proteins
(TNRC6a,b,c in mammals) (Eulalio et al., 2008; 2009; Liu et al., 2005; Zekri et al., 2009) to
mediate translational inhibition (Olsen and Ambros, 1999; Petersen et al., 2006; Pillai, 2005) and
to recruit additional factors that trigger mRNA degradation (Bagga et al., 2005; Behm-Ansmant
et al., 2006; Fabian et al., 2009; Lim et al., 2005; Rehwinkel et al., 2005). Genome-wide mass
spectrometry and mRNA expression analyses upon inhibition or overexpression of miRNAs has
revealed a modest up to ~2-fold effect on protein levels for most miRNA:target interactions
(Baek et al., 2008; Selbach et al., 2008). The majority of this effect can be attributed to changes
in mRNA levels rather than exclusive inhibition of translation (Guo et al., 2010). A flurry of
recent studies using kinetically controlled assays of miRNA activity in mouse (Fabian et al.,
2009), fly (Djuranovic et al., 2012), zebrafish (Bazzini et al., 2012), and human (Bethune et al.,
2012; Meijer et al., 2013) now present a more complete picture of the mechanism of miRNA-
mediated repression where translation inhibition occurs early on in the miRNA:target interaction
and is followed by de-adenylation, decapping, and degradation of the target transcript, such that
mRNA effects are dominant at steady state.
miRNAs: biological function
Given the ~2-fold effects of most miRNA:target interactions and the tolerance of
heterozygous loss of function mutations for most genes (i.e. a 2-fold reduction in protein levels),
it is somewhat surprising to see such widespread conservation of miRNA targeting. However, a
multitude of specific functions have been established for individual miRNA genes in almost
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