Ribonomic analysis of human Pum1 reveals cis-trans conservation across species despite ...

45 Pages·2008·1.37 MB·English

MCB Accepts, published online ahead of print on 14 April 2008
Mol. Cell. Biol. doi:10.1128/MCB.00155-08
Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Morris et al- Ribonomic analysis of human Pum1

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3 Ribonomic analysis of human Pum1 reveals cis-trans conservation D
4 across species despite evolution of diverse mRNA target seEts
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5 Running Title: Ribonomic analysis of human Pum1 D
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6 Adam R Morris, Neelanjan Mukherjee, and Jack D. Keene* loa
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8 Department of Molecular Genetics & Microbiology :/
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9 Duke University Medical Center s
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10 Durham, North Carolina 27710 /
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12 Materials and Methods word count- 894 0
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13 Introduction, Results, and Discussion Word Count- 5138 b
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* Box 3020 Research Drive, Duke University Medical Center, Durham, NC, 27710, Tel # [phone],
Fax # [phone], [email protected]
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Morris et al- Ribonomic analysis of human Pum1
1 Abstract
2 PUF family proteins are among the best characterized regulatory RNA-binding
3 proteins in non-mammalian species, but relatively little is known about mRNA targets or
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4 functions of mammalian PUF proteins. In this study, we used ribonomic analysis to
5 identify and analyze mRNAs associated with ribonucleoproteins containing an E
6 endogenous human PUF protein, Pum1. Pum1 associated mRNAs were highly enriched
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7 for genes encoding proteins that function in transcriptional regulation and cell w
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8 cycle/proliferation, results consistent with the post-transcriptional RNA regulon model a
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9 and the proposed ancestral functions of PUF proteins in stem cell biology. Analysis of f
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10 3’UTR sequences of Pum1 associated mRNAs revealed a core Pum1 consensus sequence, h
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11 UGUAHAUA. CPum1 knockdown demonstrated that Pum1 enhances decay of associated /m
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12 mRNAs, and re-localization of Pum1 to stress granules suggested that Pum1 functions in .a
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13 repression of translation. This study is the first in vivo genome-wide mRNA target o
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14 identification of a mammalian PUF protein and provides direct evidence that human PUF n

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15 proteins regulate stability of associated mRNAs. Comparison of Pum1 associated r
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16 mRNAs to mRNA targets of PUF proteins from S. cerevisiae and Drosophila ,
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17 demonstrates how a well conserved RNA-binding domain and cognate cis-regulatory 9

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18 module have been evolutionarily rewired to regulate the collective expression of different u
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19 sets of functionally related genes.
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Morris et al- Ribonomic analysis of human Pum1
1 Introduction
2 PUF family RNA Binding Proteins (RBP)s are among the best characterized
3 regulators of post-transcriptional gene expression in non-mammalian eukaryotes. Named
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4 for the founding members Pumilio (32) and FBF (61), PUF proteins are represented
5 throughout the eukaryotic lineage (47). The common feature of PUF proteins iEs the PUF
6 Homology Domain (PUF-HD), an RNA binding domain typically consisting of eight
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7 imperfect repeats of a 32 amino acid sequence (60). The overall sequence of PUF w
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8 proteins from different species is not highly similar outside of the PUF-HD, although the a
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9 PUF-HD is incredibly well conserved (46). This extreme conservation of the PUF-HD f
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10 suggests that post-transcriptional regulation of gene expression by PUF proteins has h
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11 remained importCant throughout evolution. Although there is a growing body of /m
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12 knowledge concerning PUF proteins in non-mammalian model organisms, relatively little .a
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13 is known about the functions or mRNA targets of PUF proteins in mammals. o
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14 Genetic analyses have revealed diverse functions of PUF proteins such as embryo n

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15 patterning in Drosophila (32), germ line establishment and maintenance in Drosophila r
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16 (14, 33, 42) and C. elegans (4, 10, 30, 53, 61), and mitochondrial function in S. cerevisiae ,
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17 (16). PUF proteins have been found to function as repressors of gene expression through 9

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18 both repression of translation and enhancement of decay of target mRNAs (13, 19, 20, 41, u
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19 55). Crystal structure analysis of the PUF-HD from Drosophila Pumilio revealed that it
20 forms a crescent shape (12), with protein-RNA interactions occurring on the inner
21 concave surface and protein-protein interactions on the outer surface.
22 Genome-wide target identification of five S. cerevisiae PUF proteins, Puf 1-5 (17),
23 and the single Drosophila PUF protein, Pumilio (18), demonstrated that each protein
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Morris et al- Ribonomic analysis of human Pum1
1 binds to a specific group of functionally related mRNAs distinct from those mRNAs
2 bound by any other PUF protein (except for Puf1 and Puf2, which bind overlapping sets
3 of mRNAs). Puf3 was found to bind almost exclusively to mRNAs of nuclear-encoded
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4 mitochondrial proteins, and this binding was later found to have functional consequences
5 when it was shown that Puf3 regulates stability of target messages in a conditioEn-specific
6 manner (13) and regulates mitochondrial biogenesis and motility in S. cerevisiae (16).
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7 These experiments represent a compelling example of the post-transcriptional RNA w
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8 operon/regulon model (26, 28), demonstrating a mechanism through which expression of a
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9 functionally related genes can be coordinately regulated at the level of the mRNA. f
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10 Targets of Drosophila Pumilio also contained potential RNA regulons, most notably the h
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11 vacuolar-type ACTPase and the embryo-patterning cascade, which Pumilio mutants are /m
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12 known to disrupt (18). These studies also demonstrated for the first time that while the .a
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13 cis-trans interactions between PUF proteins and target mRNAs are similar, target o
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14 messages of PUF proteins are not conserved through evolution, at least from S. cerevisiae n

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15 to Drosophila (18). r
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16 Mammalian genomes contain two PUF genes, Pum1 and Pum2 (46), both of ,
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17 which have been studied to a limited extent. The Pum2 gene has been knocked out in 9

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18 mouse, but the only obvious phenotype was smaller testis size with no effect on fertility u
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19 (58). Several potential human Pum2 mRNA targets have been discovered by various
20 groups (15, 31, 48, 56); however the in-vivo genome-wide targets of the protein have not
21 been identified. Expression of reporter constructs containing Pum2 target 3’UTRs was
22 shown to be repressed by Pum2 overexpression, but the mechanism of repression was not
23 determined (31). Rat Pum2 was found to localize to stress granules in hippocampal
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1 neurons, with Pum2 knockdown interfering with stress granule formation and Pum2
2 overexpression inducing aggregates that co-stained with stress granule markers (52).
3 Even less is known about Pum1. The human Pum1 RNA-binding domain can bind to the
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4 Nanos Response Element, an mRNA sequence bound by Drosophila Pumilio, and has
5 been found by crystal structure to interact with RNA in a very modular fashionE, with each
6 of the eight PUF repeats directly contacting a single RNA base (54). Recombinant Pum1
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7 was shown in vitro to interact with CNOT8 protein, a member of the CCR4-NOT w
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8 deadenylase complex (20), suggesting that enhancement of target mRNA deadenylation a
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9 and decay may be a conserved mechanism of PUF protein function (57). f
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10 Due to the lack of knowledge regarding both targeting and function of h
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11 mammalian PUFC proteins, this study was undertaken to identify genome-wide mRNAs /m
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12 associated with Pum1 protein, along with mechanisms by which Pum1 regulates these .a
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13 mRNAs. By determining the genome-wide targets of a PUF protein from a third species, o
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14 human, we gained insight as to how use of a highly conserved RNA-binding domain and n

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15 cognate binding sequence has changed throughout evolution by regulating different sets r
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16 of functionally related mRNAs. ,
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18 Results u
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19 Identification of Pum1 associated mRNAs
20 In this study, we used the RNA-binding protein immunoprecipitation-Chip (RIP-
21 Chip) method to identify genome-wide mRNAs associated with ribonucleoproteins
22 (RNPs) containing Pum1 (21, 27, 50). All experiments were performed in human HeLa
23 cells, and all Pum1 IPs were performed with a goat polyclonal antibody raised against a
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1 small peptide region specific to the Pum1 protein. Negative control IPs were performed
2 with pre-immune goat serum. Before performing RIP-Chip, we first confirmed specific
3 IP of Pum1 protein from cytoplasmic lysate by IP-Western, using identical conditions
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4 except that in the final step protein was extracted from the beads and used for SDS-
5 PAGE and immunoblotting. We were able to efficiently recover Pum1 proteinE in our IP
6 reactions, and correspondingly deplete a substantial amount from the original starting
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7 material (Figure 1A). After confirmation of efficient Pum1 protein recovery, we next w
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8 sought to determine whether specific mRNAs associated with Pum1 were enriched in the a
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9 Pum1 IP pellet. We chose Cyclin B1 as a potential target due to its conservation as a f
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10 PUF target in other species (3, 40, 45, 47). RT-PCR analysis confirmed specific h
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11 enrichment of CCyclin B1 mRNA in the Pum1 IP compared to negative control (Figure /m
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12 1B). After confirming that specific mRNAs associated with Pum1 protein could be .a
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13 identified using these procedures, we next performed RIP-Chip in biological triplicate o
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14 using custom spotted cDNA microarrays. Each biological replicate consisted of a Pum1 n

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15 IP sample, a negative control IP sample, and a total RNA sample, which were each r
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16 hybridized to a separate microarray along with a common reference sample. Total RNA ,
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17 microarrays were used to identify the transcriptome of the cells from which IPs were 9

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18 performed, providing an accurate background for subsequent analyses. A probe was u
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19 considered present in the transcriptome if the signal from the spot was at least two-fold
20 greater than local background in all three total RNA or Pum1 IP microarrays. T-scores,
21 based on the T-statistic, for Pum1 IP versus negative IP were calculated for all probes on
22 the microarrays (49). A visual inspection of the T-score values of the probes (Figure 2,
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1 histogram) suggested two distributions; a background distribution and a Pum1 associated
2 distribution.
3 In order to objectively define Pum1 target mRNAs based on a distribution of T-
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4 scores we employed Gaussian mixture modeling (GMM). GMM uses probabilistic
5 modeling to identify single Gaussian distributions in a population consisting ofE a mixture
6 of multiple Gaussian distributions (43). GMM uses expectation maximization modeling
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7 which is prone to converging on a local optimum, therefore several iterations of mixture w
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8 modeling were performed which initialized at different values in the distribution, and the a
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9 model with the greatest probability was then used to define Pum1-associated mRNAs. f
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10 Both Pum1 and background distributions were defined as an equation relating T-scores to h
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11 probability (FiguCre 2, curves), and using these equations we calculated the log of odds /m
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12 (LOD) of each probe being in the Pum1 distribution versus the background distribution. .a
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13 Those probes with a greater likelihood of being in the Pum1 distribution (LOD>0) were o
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14 considered Pum1 associated mRNAs (targets). While one might expect that this cutoff n

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15 would result in a high false positive rate, downstream analysis proved that a cutoff of r
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16 LOD>0 was appropriate. The list of probes, T-scores, and LOD scores is available as ,
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17 Supplementary Table 1. Use of GMM and creation of LOD scores allowed us to 9

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18 objectively define probabilities of probes being in the Pum1 distribution versus the u
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19 background distribution, and thus allowed for a less arbitrary determination of probes that
20 could be considered Pum1 associated. Probes were collapsed into unique genes, and of
21 the 6539 unique genes represented in total, 726 (11.1%) were considered Pum1 targets on
22 this basis.
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Morris et al- Ribonomic analysis of human Pum1
1 We used RT-QPCR to confirm select targets by measuring their enrichment in the
2 Pum1 IP versus either the negative IP or total RNA, using the non-target message β2M
3 for normalization and GAPDH mRNA as a negative control. QPCR analysis confirmed
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4 levels of enrichment up to 240 fold for target messages in the Pum1 IP, with levels of
5 enrichment being similar when compared to either the negative IP or total RNAE (Figure
6 3). Target messages confirmed by RT-QPCR represented a range of LOD scores greater
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7 than 0, thus the confirmation of these targets serves as partial validation of the LOD>0 w
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8 cutoff when defining Pum1 associated mRNAs, as noted above. a
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10 Pum1 Target mRNA Analysis h
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11 After defCining mRNAs associated with the Pum1 RBP, we proceeded to /m
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12 determine if the proteins encoded by Pum1 target mRNAs were functionally related, as is .a
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13 predicted by the RNA regulon model (26, 28). We analyzed Pum1 target genes using o
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14 two web-based programs, Panther (37) and WebGestalt (62), which search gene lists for n

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15 significant enrichment in gene ontology (GO) categories and other functional groupings. r
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16 We also analyzed Pum1 targets using Gene Set Enrichment Analysis (GSEA), which ,
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17 compares a rank-ordered gene list of interest to other gene sets in the GSEA Molecular 9

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18 Signatures Database to discover significant correlations between sets of genes (49). u
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19 Results from WebGestalt and Panther, as seen in Figure 4, were very similar. The
20 Panther program searches for both positively and negatively enriched categories and
21 applies a strict Bonferroni correction for multiple testing when calculating significance
22 (37), thus fewer categories were found to be enriched in the Panther analysis and the
23 statistical significance of that enrichment was lower. WebGestalt makes no correction
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Morris et al- Ribonomic analysis of human Pum1
1 for multiple testing, thus the results obtained by this method, while having lower p-values,
2 may be less biologically significant. However, results obtained using either WebGestalt
3 or Panther were in agreement. One of the more striking results of both the WebGestalt
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4 and Panther analyses was the large number of target genes and extreme significance of
5 enrichment of GO categories involved in transcription and regulation of transcrEiption.
6 Another noteworthy result was the enrichment of mRNAs representing genes involved in
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7 regulation of cell cycle and cell proliferation and differentiation, a result consistent with w
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8 proposed ancestral functions of PUF proteins in stem cell biology (57). GSEA analysis a
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9 of the Pum1 IP rank-ordered gene list created from LOD scores revealed a high degree of f
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10 correlation with various gene sets, including gene sets whose mRNAs levels were found h
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11 to decrease afterC UVC and UVB exposure, whose mRNAs increased after CMV /m
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12 infections, a gene set consisting of HOX genes, and gene sets related to cell cycle (Figure .a
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13 4C). This result provides further support for a role for Pum1 protein in stem cell function, o
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14 as well as a role for Pum1 in response to stress (see below). n

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15 Within the set of Pum1 target genes are several specific functional relationships r
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16 that represent putative RNA operons/regulons, such as that between Cyclin B1, Cdc2, ,
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17 p21 and Wee1. The Cyclin B1-Cdc2 complex is a key regulator of the G2/M transition 9

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18 of mitotic cell cycle, with p21 and Wee1 acting as negative regulators of Cdc2 u
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19 (www.biocarta.com). Although it may seem counter-intuitive that Pum1 would regulate
20 expression of both one protein and a second protein that negatively regulates the first
21 protein, this situation has been seen in C. elegans, where a PUF protein represses a MAP
22 kinase and a gene that inactivates the same MAP kinase, thereby ensuring continued
23 repression of the MAP kinase gene after PUF mediated repression of both proteins is
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1 relieved (31). PCNA, GSK3β, p21, and p27 form another cell cycle related functional
2 group, as all of these proteins function as inhibitors of Cyclin D (www.biocarta.com).
3 One of the most striking potential Pum1 regulons is that of the E2F transcription factors:
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4 four of the five E2Fs that were represented in the total mRNA population were found to
5 be Pum1 targets (E2Fs 3-6 are targets, E2F1 not a target), showing an overlap oEf two
6 highly enriched categories in Pum1 targets; cell cycle/proliferation and transcription (11).
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7 A large number of RNA processing and RNA binding protein genes were also found to w
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8 be Pum1 targets, among them the histone Stem Loop Binding Protein (SLBP), DICER, a
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9 Pum1 itself, and the other human PUF protein, Pum2. f
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11 Identification oCf the Pum1 Untranslated Sequence Element for Regulation (USER) /m
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12 Previous analyses of PUF protein target mRNAs revealed a consensus sequence .a
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13 present in the 3’untranslated regions (UTRs) of target messages (17, 18). We used the o
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14 motif finding program MEME (5) to search for a consensus sequence in the 3’ UTRs of n

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15 Pum1 targets. The 3’UTR sequences of the top 100 Pum1 associated mRNAs, by LOD r
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16 score, were used as a training set for MEME analysis, resulting in discovery of the ,
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17 consensus sequence shown in the inset in Figure 5. Contained in the Pum1 target 9

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18 consensus sequence is the eight nucleotide core sequence UGUAHAUA, which has been u
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19 shown by X-ray crystallographic analysis to be directly bound by the Pum1 PUF-HD (54).
20 We searched for the occurrence of this core sequence in the 3’ UTRs of the Pum1 targets
21 not used in the MEME training set and found it occurred at least once in 46.5% of Pum1
22 target 3’UTRs but only in 13.5% of total mRNA 3’UTRs. To determine the likelihood of
23 this enrichment occurring by chance, we created 50,000 sets of random 3’ UTRs and
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