Resource

Global Analysis of mRNA Localization

Reveals a Prominent Role in Organizing
Cellular Architecture and Function
Eric Lécuyer,1,3 Hideki Yoshida,1,3 Neela Parthasarathy,1,2,3 Christina Alm,1,2,3 Tomas Babak,1,2,3
Tanja Cerovina,1,3 Timothy R. Hughes,1,2,3 Pavel Tomancak,4 and Henry M. Krause1,2,3,*
1Banting and Best Department of Medical Research
2Department of Medical Genetics and Microbiology
3Terrence Donnelly Centre for Cellular and Biomolecular Research

University of Toronto,Toronto, Canada

4Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

*Correspondence: [email protected]
DOI 10.1016/j.cell.2007.08.003

SUMMARY More recently, it has been shown that protein localization

can also be controlled by localizing the mRNA transcript
Although subcellular mRNA trafficking has prior to translation (Bashirullah et al., 1998; Czaplinski
been demonstrated as a mechanism to con- and Singer, 2006; Kloc et al., 2002; St Johnston, 2005).
trol protein distribution, it is generally believed A potential advantage of this mechanism is its cost effec-
that most protein localization occurs subse- tiveness. Each localized mRNA can facilitate many rounds
quent to translation. To address this point, we of protein synthesis, thereby avoiding the significant en-
ergy costs of moving each protein molecule individually
developed and employed a high-resolution
(Jansen, 2001). This process also helps to ensure that
fluorescent in situ hybridization procedure to
proteins do not appear where their effects would be
comprehensively evaluate mRNA localization detrimental.
dynamics during early Drosophila embryogene- Localized mRNAs can serve many biological functions,
sis. Surprisingly, of the 3370 genes analyzed, including the establishment of morphogen gradients
71% of those expressed encode subcellularly (Driever and Nusslein-Volhard, 1988; Ephrussi et al.,
localized mRNAs. Dozens of new and striking 1991; Gavis and Lehmann, 1992), the segregation of
localization patterns were observed, implying cell-fate determinants (Broadus et al., 1998; Gore et al.,
an equivalent variety of localization mecha- 2005; Hughes et al., 2004; Li et al., 1997; Long et al.,
nisms. Tight correlations between mRNA distri- 1997; Melton, 1987; Neuman-Silberberg and Schupbach,
bution and subsequent protein localization and 1993; Simmonds et al., 2001; Takizawa et al., 1997; Zhang
et al., 1998), and the targeting of protein synthesis to spe-
function, indicate major roles for mRNA locali-
cialized organelles or cellular domains (Adereth et al.,
zation in nucleating localized cellular machiner-
2005; Lambert and Nagy, 2002; Lawrence and Singer,
ies. A searchable web resource documenting 1986; Mingle et al., 2005; Zhang et al., 2001).
mRNA expression and localization dynamics While the list of known localized mRNAs has grown
has been established and will serve as an in- steadily over the past two decades (Bashirullah et al.,
valuable tool for dissecting localization mecha- 1998; Czaplinski and Singer, 2006; Kloc et al., 2002;
nisms and for predicting gene functions and St Johnston, 2005), the prevalence, variety and overall
interactions. importance of mRNA localization events is unknown. Pre-
vious in situ screening efforts in Drosophila have estab-
lished speculative estimates of the proportion of local-
INTRODUCTION ized mRNAs, ranging from one to ten percent (Dubowy
and Macdonald, 1998; Tomancak et al., 2002). However,
Virtually all cells are polarized, partitioning their contents the detection methods used in past studies were of
to a variety of organelles, compartments and membrane insufficient resolution to observe intricate subcellular
interfaces that execute specialized biological and regula- patterns.
tory functions. Since the discovery of the signal peptide by To assess mRNA subcellular localization dynamics on
Blobel and colleagues (Blobel and Dobberstein, 1975), the a global scale, a high-resolution fluorescent in situ hybrid-
targeting of most proteins to these various subcellular ization (FISH) procedure was developed and applied to
destinations has been thought to occur after translation. early developmental stages of Drosophila embryogenesis.

174 Cell 131, 174–187, October 5, 2007 ª2007 Elsevier Inc.

Figure 1. Embryonic Gene Expression Dynamics Revealed by High-Resolution FISH
(A and B) The optimized FISH procedure reveals localization patterns not readily discernible with traditional detection methods and enables the
unambiguous distinction of maternal and zygotic mRNA populations. Examples of patterns observed are shown for maternal Bsg25D transcripts (A),
and for zygotically expressed CG4500 and Trn-SR transcripts (B), detected using optimized FISH (mRNAs in green/nuclei in red), or standard
alkaline phosphatase-based detection ([A] left panel, image obtained from the BDGP in situ database, Tomancak et al., 2002).
(C) General summary of observed and projected gene expression and mRNA localization events.
(D) Comparison of maternal and zygotic transcripts and their respective gene ontology (GO) term enrichments.
(E) Expression and localization dynamics of maternal and zygotic transcripts during stages 1–9 of embryogenesis.

Of the genes expressed during this developmental signals due to cross-hybridization to mRNAs with similar
window, a surprising 71% were found to encode mRNAs sequences is highly unlikely (Figure S2).
exhibiting clear subcellular distribution patterns. The fre- Following high-throughput FISH, samples were
quency and variety of localization events suggests that vir- mounted and analyzed using epifluorescence micros-
tually all aspects of cellular function are impacted by RNA copy. For each expressed gene, representative low and
trafficking pathways. We conclude that mRNA localization high magnification images were captured at key develop-
is a major mechanism for controlling cellular architecture mental stages and incorporated within a relational data-
and function. base. The first 4.5 hr of Drosophila development, spanning
embryonic stages 1–9, was chosen for analysis, as this in-
RESULTS terval is manageable in terms of data annotation and en-
compasses major developmental landmarks such as the
Method Development, Screening, midblastula transition (MBT), gastrulation and the specifi-
and Localization Database cation of many cell types. The MBT is the period during
To circumvent the deficiencies of existing in situ hybridiza- which developmental regulation switches from control
tion protocols, considerable effort was made to develop by maternally synthesized gene products to control by zy-
a procedure with optimal subcellular resolution, sensitiv- gotically transcribed genes (Tadros et al., 2007b). Impor-
ity, consistency, throughput and economy. Typical results tantly, our FISH method enables the unambiguous distinc-
obtained with the resulting method (Lécuyer et al., 2007), tion between maternal and zygotic mRNA populations.
versus traditional alkaline phosphatase-based probe de- Maternally provided transcripts are generally cytoplasmic,
tection, are illustrated in Figures 1A and S1 in the Supple- exhibit FISH signal intensities above background in stage
mental Data available with this article online. Reassuringly, 1 embryos and decrease in intensity during later stages.
control analyses using probes with increasing sequence Zygotic mRNAs, on the other hand, are always first
divergence indicate that the occurrence of false positive detected in nascent transcript foci within subsets of

Cell 131, 174–187, October 5, 2007 ª2007 Elsevier Inc. 175

embryonic nuclei and are not generally observed until S1), consistent with the rapid patterning and morphoge-
stage 4. Examples of maternal and zygotic mRNAs are netic processes that follow the MBT. These functional en-
illustrated in Figures 1A and 1B. richments are in agreement with a recent study by De
An annotation term hierarchy has been created to doc- Renzis et al. (2007), who identified 1158 putative zygoti-
ument stage-specific expression, localization and degra- cally expressed genes using microarray analyses of chro-
dation dynamics of each transcript. In addition, an RNA lo- mosome deletion mutants (De Renzis et al., 2007). How-
calization database resource, containing annotation terms ever, their dataset does not include 337 of the 504
and representative images of all transcripts detected, has genes that we unambiguously found to be zygotically
been established using previously described web-based expressed. Thus, by extrapolation, we predict the number
tools (Tomancak et al., 2002) and can be accessed of zygotically expressed genes during this period to be
through a searchable web-browser at: http://fly-fish.ccbr. significantly higher (2043 versus 1158; Figure 1C). Alto-
utoronto.ca. gether, our projections suggest that >9000 genes are
expressed either maternally or zygotically during the early
Embryonic Gene Expression Dynamics stages of Drosophila embryogenesis (Figure 1C), repre-
Of the 3370 genes (25% of genome) successfully senting a vast and complex set of regulatory interactions.
screened, 2314 were expressed during the developmental
window analyzed (Figure 1C). Of these, 2198 were mater- Transcript Localization
nally provided, 504 were zygotically expressed, and 388 Of the 2314 mRNAs expressed, a remarkable 71% are
were expressed both maternally and zygotically (Figures subcellularly localized (Figure 1C), with a peak in localiza-
1C and 1D). The percentage of genes that we find to be tion frequency observed between embryonic stages 4–7
maternally expressed (65%) is more than twice that of ear- (Figure 1E). This peak in localized transcript numbers
lier predictions (30%; Arbeitman et al., 2002), although it may reflect a relatively high demand for localization events
more closely resembles estimates from a recent microar- during the conversion from syncytial to cellular environ-
ray-based study (55%; Tadros et al., 2007a). Our higher ments. Alternatively, it may be skewed by the relative
values likely reflect the thorough gene-by-gene approach ease of detection of localization in large embryos versus
that was used and the improved sensitivity of the de- small cells. Further analyses of transcript localization in
tection procedure. Consistent with the importance of other tissues and developmental stages, and using higher
transcript degradation in the transition from maternal to resolution microscopy techniques, will undoubtedly reveal
zygotic control of embryogenesis (Tadros et al., 2007a), many additional localization events. Hence, our numbers
the majority (65%) of these maternal mRNAs are no represent a conservative estimate of the total number of
longer detectable by stages 8–9 (Figure 1E). localized mRNAs encoded in the fly genome.
Gene ontology (GO) term enrichment analysis reveals The 1644 localized transcripts observed were grouped
that maternal transcripts are enriched for genes involved into 35 localization categories, most of which are listed
in RNA metabolism (Figures 1D, S3, S4A, and Table S1), in Table 1. The following sections will focus on some of
including, for example, many components of the spliceo- the more diverse and striking of these localization patterns
some (crn, prp8, SmD3, snRNP69D, snRNP70K, U2af50). and their functional implications.
This finding is consistent with the large and diverse mater-
nal mRNA contingent, and the need to organize aspects of Subembryonic Localization Patterns
their processing, translation, stability and localization. In- The most prevalent localization patterns observed are the
terestingly, these terms are more strongly enriched among sub-embryonic ‘exclusionary’ categories, where mRNAs
the more stable maternal mRNA subsets that continue to be are excluded from parts of the embryo, such as the pe-
detected through stages 6–9 (Figure S4A and Table S1), in ripheral apical cytoplasm or the germline pole cells that
agreement with recent observations (Tadros et al., 2007a). form at the posterior tip (Table 1). Although these patterns
The first major burst of zygotic transcription occurs at might easily be missed in smaller somatic cells, their im-
stages 4–5 as the Drosophila embryo begins to transition portance is clear. For example, the pole cell-excluded
from syncytial to cellular growth (Figure 1E). Approxi- subgroup is specifically enriched for transcripts encoding
mately half (230) of the zygotically expressed genes de- ribosomal constituents and factors involved in mRNA me-
tected are initiated during stages 4–5 and continue to be tabolism and processing (Table S2). This is consistent with
expressed throughout the developmental period analyzed the general need to prevent transcript synthesis and trans-
(Figure S5A). Interestingly, several of the earliest tran- lation in germline cells during early embryogenesis (Sey-
scribed mRNAs, which were unexpectedly detected as doux and Dunn, 1997; Van Doren et al., 1998), and pro-
early as stages 1–2, are encoded by transposable/repeti- vides new insights into the mechanisms responsible.
tive elements (copia, Doc, Ste12DOR). The significance of Similar mechanisms are likely to be used in somatic cells
these early expression events will be addressed further to prevent translation or related processes in portions of
below. As a group, the zygotically expressed genes are the cytoplasm.
strongly enriched for functions relating to transcriptional Perhaps the most easily detected of the subembry-
regulation, cell fate determination, tissue/organ develop- onic patterns are the previously documented anterior and
ment and morphogenesis (Figures 1D, S4B, and Table posterior categories. Notably, the number of anterior

176 Cell 131, 174–187, October 5, 2007 ª2007 Elsevier Inc.

Table 1. Summary of mRNA Localization Patterns

Number of Genesa Percent of Number of Genes/Stage Range

Localization Patterns (Projectedb) Expressed St. 1–3 St. 4–5 St. 6–7 St. 8–9
Expressed 2314 (9379) 100.0 2213 2183 1592 1154
Localized 1644 (6663) 71.0 233 1578 1135 187
Subembryonic patterns 1468 (5950) 63.4 207 1431 1041 147
Exclusionary patterns 1397 (5662) 60.3 123 1371 976 8
Pole plasm/Pole cell exclusion 1272 (5156) 54.9 83 1235 920
Apical exclusion 1145 (4641) 49.5 NA 1142 747 6
Basal exclusion 256 (1038) 11.1 NA 225 112
Yolk cortex exclusion 207 (839) 8.9 58 175 6
Yolk cortex localization 277 (1123) 12.0 9 171 172 3
Posterior localization 198 (803) 8.6 74 195 106
Pole/Germ cell localization 195 (790) 8.4 NA 192 105 62
Pole plasm localization 70 (284) 3.0 70 NA NA NA
Pole buds 54 (219) 2.3 54 NA NA NA
RNA islands 44 (178) 1.9 44 NA NA NA
Anterior localization 5 (20) 0.2 5 3
Subcellular localization 366 (1483) 15.8 42 135 270 107
patterns
Basal localization 209 (847) 9.0 NA 25 190 50
Nuclei-associated localization 82 (332) 3.5 27 43 36 24
Perinuclear localization 81 (328) 3.5 25 37 35 24
Perinuclear yolk nuclei 59 (239) 2.5 6 25 35 22
Perinuclear cortical nuclei 27 (109) 1.2 8 21 2
Intranuclear accumulation 14 (57) 0.6 2 14 4 4
Apical localization 78 (316) 3.4 NA 59 54 15
Diffuse apical localization 36 (146) 1.6 NA 29 18 12
Discrete apical foci 27 (109) 1.2 NA 20 18 2
Apical clusters 24 (97) 1.0 NA 15 20 0
Apical in neuroblasts 6 (24) 0.3 NA NA NA 6
Cytoplasmic foci 45 (182) 1.9 14 38 14 15
Cell division apparatus 33 (134) 1.4 16 29 1 1
Microtubule-associated 10 (41) 0.4 9 6 1
Spindle midzone localization 10 (41) 0.4 1 10
Centrosomal localization 6 (24) 0.3 6 4 1 1
Chromatin-associated 14 (57) 0.6 2 12
Cell junction-associated 12 (49) 0.5 4 10 9 6
Membrane-associated 8 (32) 0.3 5 5 5 2
Polar body-associated 4 (16) 0.2 4 NA NA NA
NA, not applicable to these embryonic stages; St., stages.
a
All screened genes encoding localized mRNAs in either of the analyzed stages; patterns are not necessarily mutually exclusive.
b
Projected number of localized transcripts encoded in the Drosophila genome, out of a total of 13,659 coding genes.

Cell 131, 174–187, October 5, 2007 ª2007 Elsevier Inc. 177

Figure 2. Anterior/Posterior Patterns and Functional Enrichments
(A–E) Sagittal views of entire embryos (A and B) or of the posterior region (C–E) between stages 2–5, following FISH with probes to bcd (A), asp (B), osk
(C), orb (D), or grp (E) transcripts (mRNA green/nuclei red). (A and B) Varieties of anterior patterns, with bcd mRNA (A) showing tight anterior local-
ization and asp transcripts (B), a more diffuse anterior enrichment. (C–E) Early and late posterior localization patterns. While both osk and orb tran-
scripts localize to the posterior pole plasm in stage 1–2 embryos ([C and D] arrowheads), orb mRNA forms distinctive rings around pole cell nuclei at
stage 3 ([D] arrow). In contrast, grp transcripts localize in the posterior yolk plasm in early stage 4 embryos ([E] arrow). All of these transcripts localize to
the pole cells at stage 5.
(F) GO term enrichments exhibited by transcripts found within annotation categories pertaining to anterior and posterior localization in stage 1–5
embryos (column categories 1 and 2 refer to stages 1–3 and 4–5, respectively). The ‘‘hot metal’’ color scale reflects statistical significance (log 10
of the p value) of the GO term enrichments.

transcripts is far outnumbered by those that are posteri- The posterior group of mRNAs could be clearly subdi-
orly localized (5 versus 198; Table 1). For both of these vided into three categories: (1) transcripts that localize to
categories, the sensitivity and resolution of detection early pole plasm (Figure 2C), a specialized region of cyto-
enabled the identification of distinct subgroups of patterns plasm that directs the formation of germline pole cells; (2)
(Figure 2). For example, among the anteriorly localized transcripts that reside in the pole plasm and then localize
mRNAs, which include bcd, CycB, lok, milt and asp, only further into distinctive rings around pole cell nuclei
bcd exhibits tight anterior localization (Figure 2A), con- (Figure 2D); and (3) transcripts that only begin to localize
sistent with its extensively characterized function as the posteriorly in early stage 4 embryos (Figure 2E). The bio-
primary determinant of anterior cell fate specification logical significance of these subcategories is under-
(Ephrussi and St Johnston, 2004). The other four mRNAs scored by their specific GO term enrichments (Figure 2F
exhibit a more diffuse gradient of anterior enrichment (Fig- and Table S3). For example, the category 1 and 2 pole
ure 2B). Interestingly, three of these encode proteins plasm localized transcripts, which include mRNAs such
involved in cytoskeleton organization and microtubule- as aret, eIF5, gcl, Imp, nos, osk, orb, pAbp, pum, spir,
based processes (Figure 2F and Table S3). and Tm1, are strongly enriched for cell development,

178 Cell 131, 174–187, October 5, 2007 ª2007 Elsevier Inc.

Figure 3. Varieties of Apicobasal Localization Patterns and Their Functional Enrichments
(A–L) Sagittal views through the embryonic epithelium of embryos hybridized with the indicated probes. Several distinctive subcategories of apical
(A–E), basal (G–I), or exclusionary (J and K) patterns are shown. (F and L) CG14896 transcripts are apical in posterior epithelial cells (F) and in later
arising neuroblasts ([L] arrowheads). For all images, mRNAs are green and nuclei red.
(M) GO term enrichments observed for different subcategories of apical mRNAs. Enrichment scores are depicted using a hot metal color scale con-
veying statistical significance (log 10 of the p value). Column categories 2–4 refer to embryonic stages 4–5, 6–7, and 8–9, respectively.

translation regulation, pole plasm assembly and RNA Subcellular Categories: Apicobasal Patterns
localization functions (Figure 2F). In contrast, the late Besides the subembryonic localization patterns, a large
posterior group, which includes genes such as aur, collection of mRNAs, either of maternal or zygotic origin,
CG14030, CycA, grp, gwl, Rbp-1, Su(var)3-9, and ttk, were found to exhibit intricate subcellular localization
is enriched for protein kinases and negative regulators patterns. Classic examples include the subset of mRNAs
of gene expression, again consistent with previous find- that localize to the apical cytoplasm within the embryonic
ings that germ cells are transcriptionally silent (Seydoux epithelium (Figure 3). Although apical mRNAs have been
and Braun, 2006; Seydoux and Dunn, 1997; Van Doren characterized previously and considered as a homoge-
et al., 1998). Taken together, these observations sug- neous group (Davis and Ish-Horowicz, 1991; Simmonds
gest the existence of distinct early and late pathways for et al., 2001; Tepass et al., 1990), many distinctive sub-
posterior transcript localization. groups of apical transcripts could be distinguished, rang-
Notably, no maternal transcripts were identified as be- ing from broad gradients of apical enrichment to tightly
ing either dorsally or ventrally localized. Instead, differen- localized clusters or foci (Figures 3A–3E). Likewise,
tial distribution of transcripts along the dorso-ventral axis a large number of basally localized mRNAs were identi-
was always a consequence of localized zygotic tran- fied, which also fall into a number of subgroups (Figures
scription. The preponderance of transcripts localized to 3G–3I). Other patterns that vary along the apico-basal
the posterior pole of the embryo, in comparison to the axis include transcripts that are excluded from the apical
other embryonic poles, seemingly reflects special re- cytoplasm or from the entire blastoderm layer (Figures 3J
quirements for germ cell specification and the sufficiency and 3K).
of existing zygotic mechanisms to define the other coor- The functional relevance of these subgroup classifica-
dinates. tions is underlined by the GO term enrichments observed.

Cell 131, 174–187, October 5, 2007 ª2007 Elsevier Inc. 179

 Figure 4. Membrane-Associated Patterns
(A–F) Surface plane (upper panels) and sagittal
(lower panels) views of stage 3 (A and B), 4 (C),
5 (E and F), and 6 (D) embryos hybridized with
probes for the transcripts indicated in lower
panels (mRNA green/nuclei red). cno tran-
scripts localize within cortical polygonal net-
works ([A] arrowhead), while anillin mRNA is
first perinuclear ([B] arrowhead) and then
evolves into a cell junction type pattern (C).
(D–F) Patj, dlg1, and mira transcripts localize
at different positions along the lateral mem-
brane (arrowheads).

For example, the apical clusters category, which includes or perinuclear clouds as early as stage 3, and then evolve
mRNAs such as Ama, bib, Btk29A, crb, fra, Gp150, htl, Ptr, into polygonal mosaic networks shortly thereafter (Figures
scb, sog, and smo, is enriched for GO categories for 4A and 4C). These patterns resemble subsequent actin
plasma membrane and signaling pathway components filament distributions and dynamics and precede cell
(Figure 3M and Table S4). In contrast, the diffuse apical junction formation. Several other mRNAs encoding cell
group, which contains several pair-rule gene transcripts junction components, such as Patj (Figure 4D) and dlg1
(hairy, odd, prd, run), is enriched for functions associated (Figure 4E), localize at specific sites along the basolateral
with transcriptional regulation and pattern/axis specifica- membrane. In contrast, mira transcripts localize through-
tion. out the lateral membrane of embryonic epithelial cells
In addition to the apicobasal patterns detected in the (Figure 4F). Accordingly, this category is enriched for
embryonic epithelium, several mRNAs were observed GO terms related to cytoskeleton organization and biogen-
with asymmetric patterns in neuroblasts. This category in- esis (Table S5). These observations imply a significant
cludes transcripts such as asp, Gp150, mira, odd, pros, role for mRNA localization in the nucleation and position-
and wg, some of which have been observed previously ing of cytoskeletal networks and membrane-associated
(Broadus et al., 1998; Schuldt et al., 1998), and not sur- structures.
prisingly, show GO term enrichments for asymmetric cell
division functions (Figure 3M). We also identified mRNAs Cell Division and Nuclei-Associated Patterns
from uncharacterized genes, such CG14896, which Many of the most striking subcellular patterns observed
exhibit apical localization both in the posterior embryonic occur during nuclear or cellular division (Figure 5). These
epithelium underlying the pole cells and in neuroblasts include transcripts that localize to spindle poles, centro-
that arise later in embryogenesis (Figures 3F and 3L). somes/centrioles, astral microtubules, or along the mitotic
This example suggests the likelihood that many spindles themselves during anaphase and telophase (Fig-
localization mechanisms will operate in a variety of cell ures 5A–5H). Furthermore, several mRNAs that are zygoti-
types. cally transcribed in early stage 4 embryos concentrate
around metaphase chromosomes during mitosis and of-
Membrane-Associated Patterns ten become associated with spindle midbodies (Figure
Also remarkable are transcripts that localize to mem- 5D). Intriguingly, many of these mRNAs encode trans-
brane-associated structures prior to and following cellula- criptional regulators (dpld, nullo, odd, rib, run, stwl, Taf4).
rization (Figure 4). For example, cno and anillin mRNAs The genes in these categories show GO term enrich-
(Figures 4A and 4B) associate with the embryonic cortex ments for cell division related processes (Figure S3 and

180 Cell 131, 174–187, October 5, 2007 ª2007 Elsevier Inc.

Figure 5. Cell Division and Nuclei-Associated Transcripts
(A–P) Surface plane (A–L and O) or sagittal (M, N, and P) views of stage 1–5 embryos hybridized with the indicated probes (mRNA green/nuclei red).
(A–H) Examples of mRNAs that localize to different sections of the cell division apparatus, including spindle poles (A), microtubule networks and cen-
trosomes (B, C, E, and F), the spindle midzone (G and H), or in proximity to metaphase chromosomes (D). (I and J) Doc-element transcripts localize to
centromeric chromatin regions on polar body chromosomes (I) and during mitosis in diploid nuclei (J). (K and L) Ste12DOR transcripts localize in chro-
matin-associated foci during metaphase (K), which then become telomeric during anaphase (L). (M) roX1 RNA shows polarized enrichment in the
basal portion of blastoderm nuclei (arrowhead). (N) Bsg25D transcripts exhibit perinuclear localization around peripheral blastoderm and yolk nuclei.
(O and P) Several mRNAs exhibit nuclear retention; (O) cas transcripts are retained in groups of ventral nuclei following zygotic expression in stage 4
embryos, and (P) CG15634 mRNA is retained in nuclei situated just below the peripheral layer (arrowhead).

Table S5), implying important roles for localized mRNAs in homologous and in close proximity to the tandemly re-
the establishment, function and regulation of cell division peated Stellate gene cluster on the X chromosome, which
machineries. has been implicated in the maintenance of male fertility
Interestingly, several of the earliest zygotically ex- through an RNA interference process involving the Su(Ste)
pressed mRNAs, such as those encoded by the Doc gene cluster located on the Y chromosome (Aravin et al.,
and copia transposons, exhibit intricate chromatin-asso- 2001). Finally, roX1, a noncoding RNA involved in X chro-
ciated patterns. Indeed, Doc-element RNA localizes in mosome dosage compensation (Park et al., 2002), local-
the vicinity of centromeres, either along the ‘rosettes’ izes to the basal side of blastoderm nuclei, where the
formed by the polar body chromosomes or in dividing dip- X chromosome presumably resides (Figure 5M). As has
loid nuclei (Figures 5I and 5J). In contrast, mRNA encoded been demonstrated for roX1, these assorted DNA-associ-
by the Ste12DOR gene is found within large chromatin- ated RNAs may be functioning to help organize, establish
associated foci that localize to telomeric regions during and/or maintain chromatin domains. For Doc and Ste12-
anaphase (Figures 5K and 5L). Notably, this gene is highly DOR, the chromosome-associated RNA may be

Cell 131, 174–187, October 5, 2007 ª2007 Elsevier Inc. 181

 Figure 6. Correlations in mRNA and
Protein Distribution Patterns
(A–F) Stage 4 (B–E), 5 (A) and 9 (F) embryos hy-
bridized with probes for the indicated apical
(A), cell division-associated (B and C), mem-
brane-associated (D), and nuclear retained (E
and F) mRNAs (red signal, left panels) and co-
labeled with antibodies against the indicated
protein products (green signal, middle panels).
Overlaid mRNA and protein signals are shown
in the right panels. Nuclei are shown in blue in
the left and middle panels in (A)–(E). Arrow-
heads in (E) and (F) indicate nuclei showing
an accumulation of kuk and cas mRNAs,
respectively.

functioning in the ‘repeat-associated small interfering staining for selected transcripts, their protein products
RNA’ (rasiRNA) pathway, which acts in part to suppress or relevant markers was carried out (Figure 6). Examples
transposable element activity (Slotkin and Martienssen, are shown for mRNAs that are apically localized (Figure
2007). If so, this autoregulation would add a new dimen- 6A), cell division apparatus-associated (Figures 6B and
sion to our understanding of this process. 6C), or that reside at cell junctions (Figure 6D). In each
Finally, other nuclei-associated mRNAs were observed of these cases, mRNA localization is noted prior to the
that range from mRNAs with tight perinuclear localization appearance of protein, consistent with the view that
(Figure 5N) to those that appear to be uniformly localized transcript localization generally predetermines protein
throughout the nucleus (Figures 5O and 5P). These in- distribution at most subcellular destinations. Another in-
clude cas, CG15634, CG8552, Eip71CD, hb, Jra, kuk, triguing example is CG14438 mRNA, which appears to lo-
mfas, and scw. Interestingly, some of these are only re- calize at the level of centrioles and is found nestled within
tained within nuclei that appear to be dropping out of structures labeled with the pericentriolar marker CNN
the blastoderm layer (Figures 5P and 6E). As these nuclei (Figure 6C). These types of examples support the notion
are generally observed in pairs, and ‘nuclear fallout’ may that localized transcripts, and the proteins they encode,
be a consequence of unsuccessful nuclear divisions play central nucleation functions within the cell. These ob-
(Rothwell et al., 1998), this suggests a potential function servations further suggest that the necessary translation
of these mRNAs in nuclear migration, sorting and/or and secretory machineries are generally available at each
apoptosis. of these sites.
Interestingly, a reverse correlation exists for mRNAs
Colocalization of RNAs and Proteins that show nuclear retention, as shown for kuk and cas
In many of the cases cited above, where details are known transcripts (Figures 6E and 6F). Indeed, while Kuk protein
about protein localization or gene function, there is a strik- exhibits robust nuclear envelop localization in cortical
ing correlation between transcript localization and the nuclei where the mRNA is cytoplasmic, no protein is
patterns or functions of the encoded proteins (Table S6). observed in or around the yolk nuclei in which kuk mRNA
To further illustrate some of these relationships, double- is retained (Figure 6E). Similarly, in stage 9 neuroblasts,

182 Cell 131, 174–187, October 5, 2007 ª2007 Elsevier Inc.

Cas protein is expressed robustly in cells with diffuse Localized RNAs as Functional Components
cytoplasmic cas mRNA, but shows little or no protein ex- of RNP Complexes
pression in cells where cas mRNA is nuclear (Figure 6F). In some cases, localized mRNAs are likely to have roles in
These examples suggest that nuclear mRNA retention addition to targeting protein synthesis. For example, they
serves as a means of precisely timing or coordinating could well serve as structural or catalytic components
protein expression to related cellular processes (Brandt of RNP complexes, as seen for the many noncoding
et al., 2006; Grosskortenhaus et al., 2006; Kambadur RNA molecules found in ribosomes and spliceosomes
et al., 1998; Pilot et al., 2006). Although this mechanism (Eddy, 2001; Erdmann et al., 2001; Prasanth and Spector,
of translational control has only once been documented 2007). Indeed, a structural function for RNAs that localize
before (Prasanth et al., 2005), it may prove to be a to the vegetal pole of Xenopus oocytes has recently been
relatively common form of post-transcriptional gene uncovered (Kloc et al., 2005), and genetic studies of the
regulation. oskar gene in Drosophila have shown that protein null al-
leles yield only a subset of the phenotypic traits exhibited
by RNA nulls (Jenny et al., 2006). This may also be the
DISCUSSION
case for many other genes for which protein null alleles
are considered to be genetic knockouts. These additional
The striking diversity and frequency of localized mRNAs
roles would be consistent with the recent explosion of
observed in this study, and the numerous correlations be-
newly recognized RNA-dependent processes, and with
tween mRNA and protein distribution and function uncov-
the hypothesis that RNAs preceded proteins evolutionarily
ered, show that mRNA localization plays a far greater role
in many cellular functions (Gilbert, 1986). Taken further,
in coordinating cell physiology and anatomy than ever
the high proportion of the genome that is transcribed but
previously suspected. Over the years, mRNA localization
noncoding (Mattick, 2004; Willingham and Gingeras,
has primarily been thought to coordinate specialized bio-
2006) suggests that we may only now be looking at the
logical processes such as morphogen gradient formation
tip of an iceberg.
and asymmetric cell division (Kloc et al., 2002; St John-
ston, 2005). Our findings, however, necessitate a change
RNA Localization and Disease
in perspective, implicating mRNA localization as a means
As our data suggests that mRNA localization has a key
of regulating a vast number, if not the majority, of cell func-
role in targeting various cellular machineries, it can be
tions.
imagined that the integrity of transcript localization path-
ways will be essential for ensuring appropriate cell growth
mRNAs as Nucleators of Localized Complexes and differentiation, while also preventing cellular transfor-
The major inference of this study is that, since mRNA lo- mation and tumorigenesis. Indeed, inappropriate target-
calization generally occurs prior to encoded protein pro- ing of mRNAs would lead to aberrant protein distributions
duction, and is so pervasive in scope, it must play a major within the cell, interference with normal regulatory path-
role in the nucleation and assembly of protein complexes ways and altered complex stoichiometries. In support of
and organelles. One such example is anillin mRNA, which this general view, several of the localized mRNAs charac-
encodes an actin-interacting protein and is localized in terized in this study (ex: mira, dlg1, raps) encode well-
dynamic patterns that closely resemble actin filament known regulators of cell polarity and asymmetric cell divi-
distributions that form subsequently (Field and Alberts, sion with demonstrated tumor suppressor functions
1995; Karr and Alberts, 1986). Another example is the (Caussinus and Gonzalez, 2005; Pagliarini and Xu, 2003;
set of chromatin-associated RNAs, which may be acting Siegrist and Doe, 2005). Other examples include the
like some siRNAs to recruit chromatin remodeling com- CG6522 mRNA, which exhibits an early cell membrane-
plexes. Other organelles likely to be controlled by mRNA associated pattern. Homology searches reveal that this
localization include various subcomponents of the cell mRNA likely encodes the Drosophila ortholog of Testin,
division apparatus. An example is CG14438 mRNA, a cytoskeleton-associated LIM-domain protein that has
which localizes to centrosome-associated foci that been identified as a tumor suppressor in mice and humans
move during cell division to nestle within CNN-contain- (Drusco et al., 2005; Mueller et al., 2007). This example
ing clusters (Figure 6C). Notably, CG14438 encodes a typifies the potential usefulness of our database in identi-
protein containing 21 zinc finger domains of the C2H2 fying genes with human disease relevance. Altogether, our
type, which could readily serve as a nucleating scaf- findings force a reassessment of many cancer pathways
fold for RNA-containing centrosomal/centriolar com- for which modeling has been entirely concentrated on
plexes. Our findings are consistent with recent studies events and interactions occurring at the post-translational
suggesting a role for RNAs in the regulation of centro- level.
some dynamics (Alliegro et al., 2006; Blower et al.,
2005; Lambert and Nagy, 2002). Moreover, our study ex- Database Usage
tends these previous findings, by revealing a large collec- The accompanying mRNA localization database will im-
tion of cell-division apparatus associated mRNAs that pact many different biological disciplines. For example,
await further characterization. it will augment existing resources currently being used to

Cell 131, 174–187, October 5, 2007 ª2007 Elsevier Inc. 183

study functional and regulatory genetic relationships. This would also be consistent with suggestions that RNA-
Since our strategy provides additional levels of detail based processes have played a major role in promoting
with regards to spatio-temporal expression dynamics, this phenotypic variation and complexity in higher eukaryotes
dataset will also serve as a powerful tool for deciphering (Mattick, 2004).
and validating gene regulatory networks.
As there is a tight correlation between mRNA localiza-
EXPERIMENTAL PROCEDURES
tion and protein function, a more specific use for this re-
source will be its predictive value in assigning functions Probe Production
for uncharacterized genes. Indeed, 919 of the subcellu- The Drosophila gene collection 1 and 2 (DGC1 and 2) bacterial cDNA
larly localized mRNAs in this study are encoded by libraries (Rubin et al., 2000; Stapleton et al., 2002) were obtained as
poorly characterized genes designated only by ‘‘CG’’ num- bacterial glycerol stocks in 96-well plates. Overnight cultures served
bers. For many of these, we can now postulate a func- as templates for PCR using universal primers to amplify library cDNA
sequences containing flanking bacteriophage promoter elements
tion with significantly higher confidence due to our know-
(T7, T3, or Sp6). Following PCR product purification on filter plates
ledge of where the mRNA is localized in the cell and (Whatman Inc., Clifton, NJ, USA), fragments were concentrated by
which other mRNAs, proteins and organelles it tempo- ethanol precipitation and resuspended in nuclease-free water. Anti-
rally and spatially colocalizes with. These predictive sense RNA probes labeled with digoxigenin (DIG) were synthesized
values may also suggest new functions for well-chara- as described by Lécuyer et al. (2007). The efficiency and accuracy of
cterized genes. Thus, this approach may help identify all PCR amplifications and transcription reactions was systematically
verified by agarose gel electrophoresis after each step. Samples for
or confirm many new components of regulatory com-
which no PCR product was obtained, or that contained multiple frag-
plexes and pathways. We expect that previously un-
ments, were excluded from further analysis.
characterized macromolecular complexes will also be
discovered, many of which will be spatially and develop-
mentally regulated. Embryo Collection, Fixation, and Fluorescent In Situ
Hybridization (FISH)
Another area of research that will benefit greatly from
Wild-type Oregon R flies were maintained at 25 C and 50% humidity
this dataset will be the study of RNA cis-regulatory ele- in large plexiglass cages (60 3 60 3 60 cm). Following a 1 hr preclear-
ments and trans-acting factors that dictate the localiza- ing step, embryos were collected on fresh food plates for 4.5 hr and
tion of different mRNA subpopulations. In the past, processed for fixation and storage as described by Lécuyer et al.
these studies have been tedious and complicated, due (2007). Conditions and methods for FISH and double FISH are also
in part to the complexity of RNA regulatory elements, described in detail by Lécuyer et al. (2007). The following antibodies
trans-acting complexes and the limitations of RNA struc- were used for immunostaining: mouse anti-BicD 4C2 and mouse
anti-Crumbs Cq4 (Developmental Studies Hybridoma Bank, Iowa
ture prediction algorithms. Unlike DNA cis-elements,
City, IA, USA), rabbit anti-Anillin (Provided by Dr. Julie Brill, The Hos-
RNA regulatory elements tend to possess complex pital for Sick Children, Toronto, ON, Canada), rabbit anti-Cas (Kamba-
features of both sequence and structure. The richness dur et al., 1998), rabbit anti-CNN (Provided by Dr. Thomas Kaufman,
of the data set provided here will reveal common se- Indiana University, Bloomington, IN, USA), and rabbit anti-Kuk (Brandt
quence and/or structural elements. Together with the et al., 2006). Mouse and rabbit antisera were used at 1:10 and 1:100
growing list of Drosophila genome sequences available dilutions, respectively.
to assess sequence and structure conservation, rapid
advances in the identification of conserved cis-elements Sample Imaging Procedure
and trans-acting machineries should be possible. In Samples were analyzed on a Leica DMRA2 epifluorescence micro-
point of fact, a consensus localization motif for mRNAs scope equipped with a rotating stage and a Q-imaging Retiga EX
in the diffuse apical category has been identified using digital camera (Quorum Technologies Inc., Guelph, ON, Canada)
and Openlab imaging software (Improvision Ltd., Coventry, England).
exactly this approach (dos Santos et al., 2007). Tran-
For each positive sample, a combination of low (103) and high mag-
scripts that colocalize are also likely to share other nification (203 and 403) images were captured to document tran-
types of regulatory elements. For example, similarly lo- script dynamics. As often as possible, care was taken to capture
calized mRNAs may share common regulatory elements images with embryos in the traditional orientation, with anterior to
used to couple translation and stability control. the left and dorsal to the top. Controls probes were included in
each experiment to control for experimental variations in staining
levels. An autoexposure function was used as a semiquantitative
Future Prospects and Considerations
measure of maternal transcript abundance in stage 1–2 embryos,
An important point that remains to be confirmed is with exposure times used to categorize expression levels as strong
whether the extent and variety of mRNA localization (1–20 ms), intermediate (21–40 ms), weak (41–80 ms), or nonex-
events observed here occur in other tissues and organ- pressed (>81 ms). A maximum exposure time of 80 ms was used
isms. The amazing level of conservation between Dro- at 203 magnification, as this provided a comparative standard, in
sophila genes and developmental processes, and those particular for transcripts that are degraded over time. The autoexpo-
sure function was also used for DAPI images. Tyramide-Cy3 and
of higher organisms, suggests that these mRNA localiza-
DAPI images were false-colored in green and red, respectively, using
tion frequencies and mechanisms will also be highly con-
Openlab, as this color scheme was found to provide the best con-
served. In fact, it may have been the prior existence of trast. All overlaid images were saved as high-resolution TIF files. Fig-
these elements and machineries that made early Dro- ures were constructed using Photoshop (Adobe Systems Inc., San
sophila syncytial development an evolutionary possibility. Jose, CA, USA).

184 Cell 131, 174–187, October 5, 2007 ª2007 Elsevier Inc.

Data Annotation and Database Setup Arbeitman, M.N., Furlong, E.E., Imam, F., Johnson, E., Null, B.H.,
All of the image and annotation data were organized within a MySQL Baker, B.S., Krasnow, M.A., Scott, M.P., Davis, R.W., and White,
database using Perl-based annotation tools adapted from a previous K.P. (2002). Gene expression during the life cycle of Drosophila mela-
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stage ranges (stages 1–3, 4–5, 6–7, 8–9, >10) and saved with a numer-
Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry,
ical identifier. An annotation term hierarchy was created to document
J.M., Davis, A.P., Dolinski, K., Dwight, S.S., Eppig, J.T., et al. (2000).
mRNA localization, degradation, and zygotic expression characteris-
Gene ontology: tool for the unification of biology. The Gene Ontology
tics within each stage range. For each gene, relevant annotation terms
Consortium. Nat. Genet. 25, 25–29.
were selected and submitted to the database along with specific anno-
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vations. localization in development. Annu. Rev. Biochem. 67, 335–394.
Benjamini, Y., and Hochberg, Y. (1995). Controlling the false discovery
Computational Analysis rate: A practical and powerful approach to multiple testing. J. Roy.
The annotation data was converted into a binary matrix, containing Statist. Soc. Ser. B. Met. 57, 289–300.
genes on one axis and localization terms on the other, where the pres- Blobel, G., and Dobberstein, B. (1975). Transfer of proteins across
ence of a localization feature for a given gene was indicated numeri- membranes. I. Presence of proteolytically processed and unpro-
cally as ‘‘1,’’ while lack of a feature was annotated as ‘‘0.’’ This matrix cessed nascent immunoglobulin light chains on membrane-bound
was then used for GO term enrichment analysis. Functional GO anno- ribosomes of murine myeloma. J. Cell Biol. 67, 835–851.
tations for all genes were downloaded from Flybase (http://flybase.bio.
Blower, M.D., Nachury, M., Heald, R., and Weis, K. (2005). A Rae1-
indiana.edu/genes/lk/function/). Annotations were up-propagated us-
containing ribonucleoprotein complex is required for mitotic spindle
ing the GO hierarchy (Ashburner et al., 2000), and calculations were re-
assembly. Cell 121, 223–234.
stricted to genes that were both GO annotated and analyzed in this
study (1651 genes). The hypergeometric distribution was used to cal- Brandt, A., Papagiannouli, F., Wagner, N., Wilsch-Brauninger, M.,
culate probabilities of overlap between each localization category Braun, M., Furlong, E.E., Loserth, S., Wenzl, C., Pilot, F., Vogt, N.,
against all GO categories containing three or more genes. The Benja- et al. (2006). Developmental control of nuclear size and shape by
mini-Hochberg procedure (Benjamini and Hochberg, 1995) was used Kugelkern and Kurzkern. Curr. Biol. 16, 543–552.
to control for multiple testing by computing a P-value threshold corre- Broadus, J., Fuerstenberg, S., and Doe, C.Q. (1998). Staufen-depen-
sponding to a false discovery rate (FDR) of 0.25. Transcript subgroups dent localization of prospero mRNA contributes to neuroblast daugh-
were also analyzed independently for GO term enrichments using ter-cell fate. Nature 391, 792–795.
EASE (Hosack et al., 2003). EASE scores of less that 0.05 were consid-
Caussinus, E., and Gonzalez, C. (2005). Induction of tumor growth by
ered significant, as reported previously (Tadros et al., 2007a).
altered stem-cell asymmetric division in Drosophila melanogaster.
Nat. Genet. 37, 1125–1129.
Supplemental Data
Czaplinski, K., and Singer, R.H. (2006). Pathways for mRNA localiza-
Supplemental Data include five figures and six tables and can be found
tion in the cytoplasm. Trends Biochem. Sci. 31, 687–693.
with this article online at http://www.cell.com/cgi/content/full/131/1/
174/DC1/. Davis, I., and Ish-Horowicz, D. (1991). Apical localization of pair-rule
transcripts requires 30 sequences and limits protein diffusion in the
Drosophila blastoderm embryo. Cell 67, 927–940.
ACKNOWLEDGMENTS
De Renzis, S., Elemento, O., Tavazoie, S., and Wieschaus, E.F. (2007).
We thank J. T. Westwood and colleagues at the Canadian Drosophila Unmasking Activation of the Zygotic Genome Using Chromosomal De-
Microarray Centre for the DGC stocks, as well as J. Brill and P. Gold- letions in the Drosophila Embryo. PLoS Biol. 5, e117. 10.1371/journal.
bach for providing Anillin antisera. We thank B.J. Blencowe, H. D. Lip- pbio.0050117.
shitz, and C. A. Smibert for reviewing the manuscript prior to submis- dos Santos, G., Simmonds, A.J., and Krause, H.M. (2007). A stem-loop
sion. We acknowledge funding from the National Cancer Institute structure in the wingless transcript defines a consensus motif for apical
of Canada and the Canadian Institutes of Health Research (CIHR). RNA transport. Development, in press.
E.L. is supported by a CIHR fellowship and H.M.K. by a CIHR Senior
Scientist award. Driever, W., and Nusslein-Volhard, C. (1988). The bicoid protein deter-
mines position in the Drosophila embryo in a concentration-dependent
manner. Cell 54, 95–104.
Received: May 8, 2007
Revised: July 30, 2007 Drusco, A., Zanesi, N., Roldo, C., Trapasso, F., Farber, J.L., Fong, L.Y.,
Accepted: August 2, 2007 and Croce, C.M. (2005). Knockout mice reveal a tumor suppressor
Published: October 4, 2007 function for Testin. Proc. Natl. Acad. Sci. USA 102, 10947–10951.
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