Download Multiple RNA regulatory elements mediate distinct

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Development 119, 169-178 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
Multiple RNA regulatory elements mediate distinct steps in localization of
oskar mRNA
Jeongsil Kim-Ha*, Philippa J. Webster*, Jeffrey L. Smith and Paul M. Macdonald†
Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
*Both authors contributed equally to this work
for correspondence
†Author
SUMMARY
Pattern formation in the early development of many
organisms relies on localized cytoplasmic proteins, which
can be prelocalized as mRNAs. The Drosophila oskar
gene, required both for posterior body patterning and
germ cell determination, encodes one such mRNA.
Localization of oskar mRNA is an elaborate process
involving movement of the transcript first into the oocyte
from adjacent interconnected nurse cells and then across
the length of the oocyte to its posterior pole. We have
mapped RNA regulatory elements that direct this localization. Using a hybrid lacZ/oskar mRNA, we identify
several elements within the oskar 3 untranslated region
that affect different steps in the process: the early
movement into the oocyte, accumulation at the anterior
margin of the oocyte and finally localization to the
posterior pole. This use of multiple cis-acting elements
suggests that localization may be orchestrated in a combinatorial fashion, thereby allowing localized mRNAs
with ultimately different destinations to employ common
mechanisms for shared intermediate steps.
INTRODUCTION
analysis of the mRNA localization signals. For example, the
RNA signals may consist of multiple different elements,
each acting independently to program one movement or to
target the mRNA to one site. This type of organization might
allow the combinatorial use of different RNA elements to
program different localization pathways and destinations.
Alternatively, the complex RNA signals may be more than
just the sum of their parts, and may only be capable of functioning as an intact unit.
Drosophila serves as an excellent system in which to
study the mechanisms responsible for mRNA localization.
Localization of maternal mRNAs figures prominently in
Drosophila development as a strategy to deploy molecules
controlling patterning decisions. Consequently, extensive
genetic screens for mutants defective in embryonic body
patterning have provided not only examples of localized
maternal mRNAs, but also likely candidates for genes
encoding proteins involved in localization of these transcripts [reviewed by St. Johnston and Nüsslein-Volhard
(1992)]. The mRNA encoding the anterior body patterning
morphogen bicoid (bcd) is positioned at the anterior pole of
the egg during oogenesis (Berleth et al., 1988; Stephenson
et al., 1988; St. Johnston et al., 1989) and, when translated
early in embryogenesis, produces a bcd protein gradient
(Driever and Nüsslein-Volhard, 1988). Several patterning
mutants are specifically defective in bcd mRNA localization
(Stephenson et al., 1988; St. Johnston et al., 1989).
One general mechanism for targeting proteins to subcellular locations is mRNA localization. After synthesis in the
nucleus, some mRNAs become concentrated at specific sites
in the cytoplasm, leading to the subsequent localization of
their protein products. Numerous examples of this phenomenon have been recognized in recent years, and it is now
apparent that mRNA localization plays an important role in
cell function and diversification [reviewed by Gottlieb
(1990) and Macdonald (1992b)]. Very little is known about
mRNA localization signals, although cis-acting elements
necessary for localization have been identified in several
systems (Macdonald and Struhl, 1988; Mowry and Melton,
1992; Cheung et al., 1992; Dalby and Glover, 1992; Gavis
and Lehmann, 1992). In the few cases where characterization of these elements has progressed beyond their identification, the signals have been found to be large, covering
hundreds of nucleotides of RNA (Macdonald and Struhl,
1988; Mowry and Melton, 1992). This complexity may
reflect the nature of the mRNA signals examined, as each
directs a complex program of localization (St. Johnston et
al., 1989; Yisraeli et al., 1990). It seems likely that the RNA
signals provide binding sites for proteins involved in localization. Given the large size of the signals, multiple proteins
may be expected to bind. Some insight into how such
proteins mediate localization may be derived from a detailed
Key words: pattern formation, oskar, Drosophila oocyte
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J. Kim-Ha and others
Similarly, the posterior morphogen nanos (nos), which
appears in an opposing protein gradient (Smith et al., 1992;
Gavis and Lehmann, 1992), is initially localized as an
mRNA to the posterior pole of the embryo (Wang and
Lehmann, 1991), and mutants defective in posterior body
patterning fail to localize nos mRNA (Ephrussi et al., 1991).
Numerous additional examples of mRNA localization in
Drosophila oogenesis have been discovered in recent years
(Aït-Ahmed et al., 1987; Suter et al., 1989; Kim-Ha et al.,
1991; Ephrussi et al., 1991; St. Johnston et al., 1991;
Golumbeski et al., 1991; Dalby and Glover, 1992; Cheung
et al., 1992; Macdonald, 1992a; Barker et al., 1992; Lantz
et al., 1992) and should eventually allow comparisons of
different and perhaps related mRNA localization signals and
mechanisms.
Several maternal mRNAs are localized to the posterior
pole of the developing Drosophila oocyte. The oskar gene,
required both for posterior body patterning and germ cell
determination (Lehmann and Nüsslein-Volhard, 1986),
encodes one such mRNA (Kim-Ha et al., 1991; Ephrussi et
al., 1991). Localization of osk mRNA is an elaborate process
involving movement of the transcript first into the oocyte
from adjacent interconnected nurse cells, then across the
length of the oocyte to its posterior pole. In the experiments
described here, we identify the osk mRNA localization
signal within the 3′ untranslated region (3′ UTR) of the transcript. We describe a systematic deletion analysis of the osk
mRNA localization signal, which has allowed us to identify
several elements affecting different steps in the process.
This use of multiple cis-acting elements suggests that localization may be orchestrated in a combinatorial fashion,
allowing localized mRNAs with ultimately different destinations to employ common mechanisms for shared intermediate steps.
MATERIALS AND METHODS
Fly strains
w1118 flies were used as recipients for P-element-mediated transformation. Mutant strains for analysis were the following: capu2,
spir1, stau2, osk2 and osk6 [all mutants are described in Lindsley
and Zimm (1992)].
DNA constructions
All DNA alterations were introduced by standard cloning procedures into a P-element transformation vector, pCaSpeR (Pirotta,
1988) containing a 9 kb fragment of osk previously shown to
rescue the osk mutant body patterning defects in transgenic flies
(Kim-Ha et al., 1991). In an initial set of constructs, the complete
lacZ-coding sequence was fused to the osk-coding sequence at
various positions, followed by the osk 3′ UTR. Unfortunately,
transgenic flies bearing these constructs accumulated multiple
hybrid osk/lacZ transcripts of different sizes. Northern blot
analyses using probes from different regions of the the lacZ gene
and osk 3′ UTR revealed that transcripts of less than the expected
length were truncated within the lacZ sequences, and thus did not
have the osk 3′ UTR sequences (data not shown). To avoid this
problem, all subsequent constructs tagged with lacZ carried only
the 5′ ~1.3 kb of lacZ-coding sequences, extending from a BamHI
site (positioned immediately before codon 9) to the first internal
MluI site (olc 3-5) or the first internal SspI site (olc1-2). Both sites,
which are separated by only 72 bp, were first modified by addition
of NotI linkers. In olc1 and olc5, this lacZ tag was introduced into
osk at position 2471 [all nucleotide coordinates are from Kim-Ha
et al. (1991)]. The same position defines the right (3′) deletion
endpoints of olc2, olc3, olc4 and olc6. Left (5 ′) deletion endpoints
are: olc2, 1310; olc3, 510; olc4 and olc6, 347. olc5 and olc7 are
deleted for positions 2669-3397. In olc6 and olc8, the 3′-most
portion of osk extending from position 3434 and including the
polyadenylation signal is replaced with the polyadenylation signal
of a Drosophila α-tubulin gene (Macdonald and Struhl, 1988). A
series of deletions covering the final 80 bp of the coding region
and the 3′ UTR of the osk gene, olc11-17 and olc21-29, were all
derived from olc4 using the restriction enzyme sites indicated in
Fig. 3. Nucleotide positions of these sites are as follows: StyI,
2471; EcoRI, 2669; DraI, 2793; PvuII, 2914; SphI, 3083; NdeI,
3153; SacII, 3231; PstI, 3342; EcoRI, 3397. NotI linkers were
introduced to the right deletion endpoints of olc11 and olc21-24
to allow the fragments to be joined to the NotI site inserted at the
end of the lacZ fragment. HindIII linkers were added to both the
right and left deletion endpoints of olc12-17 and olc25-29 to join
the fragments.
Transgenic animals
Transgenic fly strains were established by P-element transformation (Rubin and Spradling, 1982; Spradling and Rubin, 1982). Flies
carrying constructs to be tested for rescue of the osk mutant were
further characterized to identify the chromosome with the
transgene. Rescue of the osk− maternal-effect lethality was tested
for olc7 and olc8 by crossing into an osk6/osk6 background. Rescue
was scored by the ability of the homozygous mutant flies to
produce viable offspring.
RNA analysis
Total ovarian RNA was prepared and RNAse protection assays
were performed as previously described (Macdonald et al., 1986),
using probes that allow us to compare directly the transcript levels
of endogenous osk and each transgene. Two to six independent
lines were tested for each construct, with the exception of olc15,
for which only one line was recovered. All lines transformed with
the same construct were found to express the transgene at comparable levels.
One to three lines for each construct were assayed for localization by in situ hybridization. Transcripts were detected by wholemount in situ hybridization to ovaries from flies homozygous for
the transgene. The procedure was done according to the method of
Tautz and Pfeifle (1989) with modifications previously described
in Kim-Ha et al. (1991). The hybridization probe, an in vitro-transcribed antisense RNA complementary to the 1.3 kb lacZ insert,
was made with digoxigenin-labeled UTP and hydrolyzed by incubation in 40 mM NaHCO3, 60 mM Na2CO3 at 60°C for 90 minutes.
Samples were dehydrated in an ethanol series and mounted in
Gary's Magic Mountant (Lawrence et al., 1986) for microscopy.
Computer analysis
Sequence comparisons and RNA secondary structure analysis were
performed using the University of Wisconsin GCG programs,
Version 7.1 (Genetics Computer Group, 1991). The nucleotide
sequence of the 3′UTR of osk was compared with those of bicoid
(Berleth et al., 1988), nanos (Wang and Lehmann, 1991), cyclinB
(Whitfield et al., 1990), orb (Lantz et al., 1992), fs(1)K10 (Prost et
al., 1988), pumilio (Macdonald, 1992a), yemanucleinα (AïtAhmed et al., 1987), BicaudalD (Wharton and Struhl, 1989), tudor
(Golumbeski et al., 1991) and staufen (St. Johnston et al., 1991)
using the programs COMPARE and BESTFIT. The secondary
structure of the osk 3′UTR was analyzed using the program
FOLDRNA to predict the lowest energy structure of the entire
region and STEMLOOP to predict smaller regions of potential
secondary structure.
oskar mRNA localization elements
RESULTS
The 3 untranslated region of the osk mRNA
contains a localization signal
Within the Drosophila ovary, individual ovarioles contain a
series of progressively older egg chambers. Each egg
chamber consists of 15 nurse cells and a single oocyte, interconnected as a consequence of incomplete cytokinesis and
surrounded by somatic follicle cells. Localization of osk
mRNA occurs in a series of steps during the course of
oogenesis (Kim-Ha et al., 1991; Ephrussi et al., 1991). First,
osk mRNA is concentrated in the oocyte during early stages
of oogenesis, when the oocyte is similar in size to each of
the connected nurse cells [stages 1-6; stages are as indicated
in King (1970)]. Then, as the oocyte begins to expand
relative to the nurse cells, osk mRNA transiently accumulates at the anterior margin of the oocyte (stages 7-9). The
anterior accumulation is accompanied by the appearance of
osk mRNA at the posterior pole of the oocyte, and, as
oogenesis progresses, the anterior localization disappears
while posterior localization increases (stages 9-10). During
stage 10, osk mRNA also accumulates in the nurse cells; this
mRNA is apparently transferred into the oocyte together
with the rest of the nurse cell contents in stage 11.
Localized mRNAs typically have localization signals
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within their transcripts. To define the osk localization signal,
we generated transgenic fly strains expressing modified
forms of the osk gene. In the first, olc1 (osk localization
construct 1), the osk gene is ‘tagged’ by the addition of a
foreign DNA sequence (~1.3 kb of the coding region of the
E.coli lacZ gene). The tag allows us to follow transcripts of
the transgene by in situ hybridization without simultaneous
hybridization to the endogenous osk message. Localization
of the olc1 mRNA is identical to that of the endogenous osk
mRNA, both in oogenesis (data not shown; pattern is the
same as that of olc4, Fig. 1B) and in embryogenesis (data
not shown). To confirm that the normal mechanisms are
being used to localize the olc1 mRNA, we also monitored
its distribution in mutants defective in osk mRNA localization. In ovaries of flies homozygous for cappuccino, spire,
staufen or osk mutations, olc1 transcripts behaved like those
of the endogenous osk gene (data not shown). Therefore,
addition of the foreign sequence tag does not significantly
alter localization of the transgenic osk mRNA.
Additional osk transgenes from the initial series map the
mRNA localization signal to the 3′ UTR. olc2-olc6 are
derived from olc1 by deleting portions of osk sequences
(Fig. 1A, left panel); each was tested for mRNA localization by in situ hybridization. Progressive removal of almost
all of the osk-coding region in olc2, olc3 and olc4 has no
Fig. 1. Sequences necessary for osk mRNA
localization are located in the 3′ UTR.
(A) Constructs used to create transgenic fly
strains expressing altered forms of the osk
gene are shown in schematic form. 2,124 bp
of the coding region and 925 bp of the
3′UTR were progressively removed. The
transcribed portion of osk is shown as a
heavy line, deletions are indicated by
interruptions in the lines and the
heterologous polyadenylation signal from
tubulin is represented by a hatched box.
Foreign DNA used to tag the mRNA is ~1.3
kb of the E. coli lacZ gene, shown as an
inverted triangle at the site of insertion, near
the 3′ end of the osk-coding region.
Localization properties of the encoded
transcripts during oogenesis are indicated
by a plus or a minus; localization was
monitored by in situ hybridization for olc1olc6, and by rescue of osk− mutant flies for
olc7 and olc8. Failure to detect localized
transcripts is not due to lack of expression,
as shown in the right panel. Total ovarian
RNA from olc1, olc5 or w1118 control flies
carrying no transgene was probed for osk
transcripts by an RNAase protection assay.
The probe includes both lacZ and osk
sequences, allowing the endogenous and
transgene mRNAs to be distinguished and
compared directly; the upper band corresponds to the transgenes and does not appear in the w1118 control, while the lower band corresponds
to osk and appears in all samples. olc1 and olc5 mRNAs are present at similar levels. (B,C) Detection of transgenic mRNA distribution
patterns in ovaries by in situ hybridization. Early stage egg chambers are at left, with egg chambers of increasing age displayed rightwards.
Panel B shows the distribution of olc4 mRNA. Just as for the endogenous osk mRNA, the tagged olc4 mRNA is concentrated in the oocyte
during early stages of oogenesis (left), transiently accumulates at the anterior margin of the oocyte as it begins to expand during mid
oogenesis (center) and then becomes localized to the posterior pole of the oocyte during later stages (right). Similar results were obtained
for olc1, olc2, olc3 and olc6 mRNAs in oogenesis (data not shown). Panel C shows the results of in situ hybridization to olc5 mRNA. No
localized transcripts are detectable. Rarely, there is some weak transcript concentration within early stage oocytes.
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effect on osk localization up to at least stage 10B of
oogenesis, as shown for the most extreme example, olc4, in
Fig. 1B. Similarly, replacement of the extreme 3′ portion of
the osk mRNA in olc6 with a heterologous polyadenylation
signal also has no noticeable effect on localization. In
contrast, olc5, which lacks a 728 bp portion of the 1,043 bp
osk 3′ UTR, is severely impaired (Fig. 1C). The only vestige
of localization is weak and inconsistent concentration in
early stage oocytes; olc5 mRNA is never detected at the
posterior pole of the oocyte. This effect is not due to altered
levels of transgene expression, as olc1 and olc5 transcript
levels are similar (Fig. 1A, right panel).
Complementary results were obtained with two osk derivatives in which only the 3′ UTR was altered. The olc7 and
olc8 transgenes retain the complete osk-coding region;
function was tested by their ability to rescue the body patterning defects of an osk mutant. Again, replacement of the
extreme 3′ portion of the osk mRNA with a heterologous
polyadenylation signal has no noticeable effect on osk
function (olc8), while deletion of a large portion of the 3′
UTR eliminates the ability to rescue (olc7). Although these
functional assays provide no indication of the nature of the
defect, the olc7 result is consistent with a defect in mRNA
localization.
Distributions of the tagged transgene mRNAs for olc1,
olc4 and olc5 were also monitored in early stage embryos
(data not shown). Transcripts of olc1 are restricted to the
posterior pole, as is wild-type osk mRNA (Kim-Ha et al.,
1991; Ephrussi et al., 1991), while olc5 transcripts remain
unlocalized in embryos, as they are in ovaries. Curiously,
however, olc4 transcripts are no longer found at the
posterior, despite being localized normally in late stages of
oogenesis. This result may be related to the low yet consistent levels of olc4 expression (Fig. 2), and is considered in
more detail in the Discussion; however, as the loss of
detectable localized olc4 message apparently occurs quite
late in oogenesis, it does not affect our analysis of the cisacting elements controlling the initial localization of osk
mRNA to the posterior pole of the oocyte.
Mapping functional localization elements within
the osk 3 UTR
Additional transgenic deletion mutants were constructed to
map more precisely the mRNA localization elements within
the osk 3′ UTR (Fig. 3). All are derived from olc4, which is
expressed at low levels (Fig. 2) but displays wild-type
mRNA localization until the very late stages of oogenesis
(Fig. 1B). The low level of expression may reveal modest
defects in localization more readily than would the transgenes expressed at high levels. [Alternatively, it could be
posited that transgenes expressed at high levels would
overload the localization machinery and thereby reveal
subtle defects in localization. However, in flies with four
extra copies of the wild-type osk gene, obvious defects in
mRNA localization are only rarely observed (Smith et al.,
1992).] Among the mutants derived from olc4, olc11
through olc17 include adjacent and non-overlapping
deletions that collectively eliminate most of the osk 3′ UTR.
Mutants olc21 through olc29 have larger deletions that
extend various distances from either end of the above region.
All the deletion mutants retain the olc4 lacZ tag, allowing
Fig. 2. The olc4 transgene is expressed at a low but constant level.
Total ovarian RNA from flies carrying the indicated transgenes
was subjected to an RNAase protection assay. The probe contains
both lacZ and osk sequences, allowing a direct comparison of
transcript levels. Transgene mRNAs are detected as two bands, as
indicated, while the endogenous osk mRNA is represented by a
single band. Each independent olc4 transgene is expressed at
levels significantly and consistently lower than the endogenous
osk gene and the other transgenes from the initial set, including
olc3, which is most closely related. Notably, olc4 transcripts are
localized correctly in ovaries. All of the later series of constructs
are derived from olc4 and are expressed at similar low levels (data
not shown).
the distribution of hybrid transcripts to be followed by in
situ hybridization. The expression levels of the transgenic
constructs were determined by RNAse protection and all
were found to be similar to that of the parent construct olc4
(data not shown).
Analysis of the various mutants provides a striking result;
different regions of the osk 3′ UTR are required for different
phases of the localization process. In the sections below, we
describe each type of defective localization pattern, progressing chronologically through the normal sequence of
events.
Mutants defective in the concentration of osk
mRNA in the oocyte
Among the smaller deletion mutants, olc15 and olc16
display the earliest defects in localization, lacking the strong
concentration of osk mRNA in the oocyte during stages 16. Although some transcript accumulation in the oocyte can
be detected, it is consistently less than for olc4. Moreover,
unlike wild-type osk and olc4, transcripts of both mutants
accumulate noticeably in all nurse cells at early stages (Fig.
4A). olc15 and olc16 therefore define a localization element
or elements required for efficient movement of the transcript
from the nurse cells to the oocyte. However, these two
mutants do have somewhat different effects. At later stages
of oogenesis, olc15 shows normal posterior localization of
oskar mRNA localization elements
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Fig. 3. Mutants for mapping of the osk
localization signal and properties of their
transcripts. All mutants derive from olc4 (Fig.
1A) and differ only in the region shown, which
includes the final 80 bp of coding region (at left)
and the 3′ UTR of the osk gene. Each transcript
is represented by a heavy line, interrupted for
deletions. Three patterns in osk localization are
indicated; the first and last are normal events,
with wild-type scored as plus. Minus indicates
that the mutant is significantly impaired,
although the step may not be completely
eliminated. The middle step, prolonged anterior
accumulation, does not occur in wild-type, where
it is scored as minus. Plus indicates that this
abnormal step occurs. Note that early defects
may obscure later events.
the modest level of transcripts which do enter the oocyte; in
contrast, olc16 transcripts cannot be detected at the posterior
pole (Fig. 4A, right). It is not clear if the late defect in olc16
localization is simply a consequence of severely impaired
transport to the oocyte, or if olc16 is also specifically
defective in localization to the posterior pole of the oocyte
(see Discussion). Larger deletions that lack the olc16 region,
olc25-29, also display an early defect in localization to the
oocyte and do not support normal posterior localization (Fig.
3).
Mutants defective in osk mRNA movement
through the oocyte
Two small deletion mutants are impaired in a normal intermediate step in localization, the accumulation of osk mRNA
at the anterior margin of mid-stage oocytes. This pattern is
typically very transient and is no longer detected in stage 10
egg chambers (see Fig. 1B). In contrast, the anterior accumulation of olc17 transcripts is both enhanced and
prolonged (Fig. 4B). The defect is itself transient and olc17
mRNAs eventually become localized to the posterior pole
(Fig. 4B). A similar but less pronounced effect is observed
for olc13 (Fig. 3). The localization phenotype of both these
mutant transcripts closely resembles that of wild-type osk
mRNA in certain BicaudalD (BicD) mutants (Kim-Ha et al.,
1991; Ephrussi et al., 1991), suggesting that localization
elements disrupted by the olc17 and olc13 deletions may
mediate a step in which BicD acts.
Mutants defective in posterior localization of osk
mRNA
Mutants olc11 and olc12 do not strongly affect mRNA localization (Figs 3, 4C). However, when both regions are absent,
in olc21, posterior localization at mid and late stages is
greatly reduced. Some transcript accumulation at the
anterior margin of the oocyte is present, but posteriorly
localized transcripts can be detected only by extensive
overdevelopment of the in situ hybridization detection
reaction, and then only inconsistently. Despite this defect,
movement of olc21 mRNA into the oocyte at early stages
remains apparently normal (Fig. 4D). Thus, elements
present within the deleted regions must play a major role in
posterior localization within the oocyte, but are not required
for either transport into the oocyte or the transient accumulation at its anterior margin in stages 8-9.
Mutants lacking multiple localization elements
Most of the mutants with larger deletions lack multiple
localization elements involved in different steps. For each
of the three general examples provided by our mutants, the
results are as would be expected from the normal progression of the different steps.
The first is the combination of deletions impairing
movement to the oocyte (olc 15 and olc16) and displaying
enhanced anterior accumulation of the transcripts (olc17 and
olc13). Mutant olc25, which is effectively a combination of
olc16 and olc17, behaves like the olc16 mutant alone; transcripts are not concentrated in the oocyte. Anterior accumulation is not observed, presumably because of the low
level of transcripts within the oocyte. Additional larger
mutations encompassing both elements (olc26-olc29)
display similar phenotypes (Fig. 3).
The mutants olc22 and olc23 have deletions of a pair of
elements, which individually cause prolonged anterior accumulation (olc13) and impair posterior localization of the
message within the oocyte (olc21). Transcripts of these
mutants display the initial defect, strong anterior accumulation in mid-stage oocytes. In olc13 mutants, this defect is
transient and transcripts eventually move to the posterior
pole of the oocyte. In the larger olc22 and olc23 mutants, the
anterior accumulation of transcripts remains transient, but
there is no accompanying localization to the posterior pole.
Finally, one large mutant, olc24, lacks localization
elements involved in each of the three identified steps. Not
surprisingly, olc24 transcripts are not localized at any stage
(Fig. 3).
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Fig. 4. Transgenic mRNA distributions of representative mutants. In situ hybridizations are presented as in Fig. 1B and C. (A) olc16. This
mutant is defective in early movement into the oocyte, and does not show subsequent localization to the posterior pole. (B) olc17. This
mutant displays excessive transcript accumulation at the anterior margin of the oocyte. Similar results were obtained for olc13, as well as
for larger deletion mutants which remove either element but not the elements necessary for efficient transport from the nurse cells into the
oocyte (Fig. 3). (C,D) A demonstration of redundant elements in the olc11 and olc12 deleted regions. The transcript distribution for olc11
(C) and also for olc12 (not shown) is similar to wild-type. However, the olc21 mutant transcript (D), missing both the olc11 and the olc12
deleted regions, shows a severe defect in localization to the posterior pole of the oocyte; the prior transport from the nurse cells into the
oocyte remains apparently normal.
Apical nurse cell localization of hybrid osk
transcripts
For several of our osk hybrid gene constructs, including
olc1, olc26, olc27 and olc28, a very small fraction of the egg
chambers examined have a novel site of transcript accumulation, at apical regions of each of the nurse cells (Fig. 5A).
This pattern was not observed for the wild-type osk mRNA
in whole-mount ovary preparations (Kim-Ha et al., 1991;
Ephrussi et al., 1991), but can be detected by in situ
hybridizations to sectioned ovaries (N. Pokrywka and E.
Stephenson, personal communication). Curiously, this
pattern closely resembles a normal intermediate step in
localization of bcd mRNA to the anterior pole of the oocyte
[(Stephenson et al., 1988; St. Johnston et al., 1989); Fig.
5B]. Mutants that display this pattern all have the lacZ
sequence tag, yet not all tagged transcripts are seen to be
oskar mRNA localization elements
Fig. 5. Apical localization of olc27 (A) and bcd (B) transcripts in
nurse cells. Arrowheads indicate some of the apical regions where
the transcripts are concentrated. Note that, with extended
development of the in situ hybridization detection reaction, a very
low level of posteriorly localized olc27 mRNA can be detected
(see Discussion).
apically localized. However, given the low frequency at
which we detect this pattern, it is possible that all of our constructs behave similarly, but that the transient nature of this
localization does not make it readily detectable. Perhaps osk
mRNA normally passes very rapidly through the apical
zones of the nurse cells in the course of localization and thus
is not observed in whole-mount staining, whereas the
movement of the hybrid transcripts is somewhat slowed
down and thus can occasionally be detected.
DISCUSSION
Organization of mRNA localization elements
A variety of localized mRNAs have been identified in
Drosophila as well as in other systems. For example, actin
mRNA is preferentially concentrated at the growing margins
of cultured fibroblasts, a relatively simple localization
pattern (Lawrence and Singer, 1986). More elaborate
patterns of localization are seen for the localized maternal
mRNAs of Xenopus and Drosophila (e.g. St. Johnston et al.,
1989; Yisraeli et al., 1990; Kim-Ha et al., 1991; Ephrussi et
al., 1991). Although patterns of localization range from
simple to quite complex, all such mRNAs appear to contain
cis-acting localization signals; presumably these signals are
recognized and bound by proteins. Currently, the most
extensively characterized cis-acting localization signals are
from mRNAs with fairly elaborate localization patterns; all
cis-acting elements examined thus far occupy large
segments of RNA (Macdonald and Struhl, 1988; Mowry and
Melton, 1992), suggesting that multiple proteins bind to the
175
signals. Because very little is known about fine structure
within the large localization signals, we know little about
how the multiple steps of localization are directed. One possibility is that the localization signal acts as a single indivisible unit to specify the entire process. Alternatively,
complex localization signals might consist of multiple
separate elements, each directing a particular step in the
process. Such a modular organization could allow the combinatorial assembly of localization signals, with which
different mRNAs would be directed along common
pathways, but to different ultimate destinations. Our
analysis of the osk localization signal provides strong
support for the latter model. We find multiple distinct
elements within the osk localization signal, with different
elements responsible for different steps in the process. Furthermore, our results clearly demonstrate that at least some
localization elements act independently. This feature
supports the notion that the localization signal is indeed a
composite of protein-binding sites, which need not function
within a larger unit. Steps directed by any individual element
may be comparable to the entire localization process for
mRNAs with simple localization profiles. Consequently,
what is learned about individual localization elements from
complex localization signals may apply to the cis-acting
signals of mRNAs with simple localization patterns, and
vice versa.
RNAs frequently have functionally significant secondary
structure. While computer programs (see Materials and
Methods) predict many possible stem-loop structures
throughout the osk mRNA 3′UTR, we do not know which,
if any, of these structures actually form. Our data do not
support the idea of a single large secondary structure that is
necessary for the proper presentation of the multiple binding
sites, as we are able to delete large portions of the 3′ UTR
(for example, olc21-23) and still retain some steps in the
localization pathway.
oskar localization elements and candidate binding
factors
Genes involved in osk mRNA localization have been identified by maternal effect mutants defective in body patterning. Mutations in the genes cappuccino, spire, staufen, BicD
and even osk itself arrest or otherwise affect localization at
different stages of the process (Kim-Ha et al., 1991;
Ephrussi et al., 1991; Suter and Steward, 1991). Similarly,
we have found that the deletion of different fragments of the
osk 3′UTR affects different steps of localization. Mutations
in a gene whose product interacts with one of the cis-localization elements identified here might be expected to affect
localization of the endogenous osk mRNA in a way that
mimics the loss of the element. Below, we describe the
different localization elements that we have defined, and
discuss possible factors that might interact with each one.
The localization element(s) defined by transgenes olc15
and olc16 is defective in efficient transport of the transcript
from the nurse cells into the oocyte. For one of these mutants,
olc15, the defect is largely limited to the early stages of
oogenesis. There is substantial olc15 transcript accumulation
within later stage oocytes, such that some posteriorly
localized mRNA can be detected. Several explanations are
possible for the different behaviors of olc15 and olc16
176
J. Kim-Ha and others
mRNAs. First, olc15 may simply be less severe than olc16.
Secondly, there may be different requirements for transport
from the nurse cells to the oocyte at different stages of
oogenesis. Finally, olc16 may be deficient in two processes
- transport into the oocyte and posterior localization within
the oocyte - while olc15 is defective in only the first.
What factors might interact with the olc15/olc16
elements? Mutants of egalitarian and certain BicD alleles
do in fact block the early concentration of osk mRNA in the
oocyte (Ephrussi et al., 1991; Suter and Steward, 1991).
However, these mutations also cause an arrest of oogenesis
at a very early stage (Mohler and Wieschaus, 1986;
Schüpbach and Wieschaus, 1991), and such a general defect
makes it difficult to determine if specific interactions
between the encoded proteins and osk mRNA are likely.
A second type of localization element is defined by the
olc13 and olc17 transgenes. Deletion of either region results
in a prolonged accumulation of the transcript at the anterior
margin of the oocyte, prior to movement to the posterior
pole. This anterior accumulation is normally a very transient
intermediate in osk mRNA localization. Notably, a very
similar phenotype of osk localization is associated with particular mutations in the BicD gene (Kim-Ha et al., 1991;
Ephrussi et al., 1991). BicD encodes a putative coiled-coil
myosin-related protein (Suter et al., 1989; Wharton and
Struhl, 1989), but we do not know how BicD acts in the
localization process. The BicD protein could contact osk
mRNA directly in the regions defined by olc13 and/or olc17.
Alternatively, BicD might act indirectly, perhaps by facilitating the transition between two types of movement: from
the nurse cells to oocyte, and within the oocyte from the
anterior site of entry to the final destination at the posterior
pole. In any event, our results suggest that mutant BicD
proteins that display this phenotype are simply not performing their normal function as well as they should. Previously, these BicD mutations have been thought to confer
a novel activity on the encoded protein (Mohler and
Wieschaus, 1986). Curiously, a number of genes can be
mutated to exhibit a bicaudal phenotype and the appearance
of this phenotype is frequently sensitive to the genetic background (Bull, 1966; Nüsslein-Volhard, 1977; Mohler and
Wieschaus, 1986; Tearle and Nüsslein-Volhard, 1987). We
suggest that in some or all of these cases the phenotype
arises through modest and possibly transient defects in osk
mRNA localization, which allow osk protein to accumulate
in the anterior of the oocyte at levels sufficient to override
normal anterior development.
Mutants olc13 and olc17 produce a similar phenotype of
enhanced anterior accumulation, yet have deletions distant
from one another. This behavior might arise if both regions
were involved in a shared secondary structure. Computer
analysis predicts a double-stranded ‘stem’ which could form
between bases 2901-2910 and 3373-3382, contained within
the sequence elements defined by olc13 and olc17, respectively; the validity of this prediction in vivo is unknown.
Alternatively, the olc13 and olc17 regions may contain
separate binding sites for the same localization protein, presumably a protein that must interact with multiple sites for
full function.
A third localization element is defined by olc21, which is
specifically defective in posterior localization within the
oocyte. A trans-acting factor that might mediate the role of
this region is stau (St. Johnston et al., 1991). Both stau
mutants and olc21 result in an accumulation of mRNA at
the anterior margin of the oocyte, although the stau effect is
much more prominent (Kim-Ha et al., 1991; Ephrussi et al.,
1991). In both cases, the accumulated mRNA eventually
disperses, but does not continue to the posterior pole of the
oocyte (as it does in olc13, olc17 or BicD mutants). The
olc21 region clearly contains more than one localization
element, as it can be deleted in two parts with no significant
effect on localization of the transgenic mRNAs (olc11 and
olc12). Redundancy can be invoked to explain this observation; redundant binding sites for the same localization
protein may lie in each mRNA segment, or redundant mechanisms may employ different binding sites in adjacent
regions of the mRNA.
Wild-type osk mRNA must be actively maintained at the
posterior pole of the oocyte during late stages of oogenesis.
Such maintenance depends upon osk protein; in certain osk
mutants, osk mRNA diffuses away from the posterior pole
during late oogenesis and no localized transcripts can be
detected in the embryo (Kim-Ha et al., 1991; Ephrussi et al.,
1991). As described in the Results, transcripts of olc4 (upon
which all other deletion constructs are based) are not
detected at the posterior pole of early embryos. It is possible
that olc4 lacks some of the sequences that mediate the osk
protein-dependent maintenance of the mRNA at the
posterior pole; if so, it cannot be lacking all of the elements
required for this maintenance, as olc4 transcripts remain
localized until at least stage 10B of oogenesis while, in the
most extreme osk mutant, localization is lost by stage 9
(Kim-Ha et al., 1991; Ephrussi et al., 1991). Alternatively,
little is known about the turnover rates and relative levels of
localized osk message throughout oogenesis and early
embryogenesis, and it is possible that the low starting levels
of olc4 transcript are simply insufficient to be detected by
early embryogenesis.
It is notable that the assay used here to monitor localization, detection of a tagged transgenic mRNA by in situ
hybridization, does not readily allow us to detect subtle differences in the levels of localized transcripts. Consequently,
modest defects in localization will have been missed; for
example, it is possible that both olc11 and olc12 do in fact
have slight defects, but it is only when both deletions are
combined in olc21 that we are able to detect a phenotype.
We also note that the possibilities for redundancies in localization elements or mechanisms are substantial. In many of
the deletion mutants that are defective in either transport into
the oocyte or posterior localization, very low levels of
correctly localized mRNAs can be detected by increasing
the sensitivity of the in situ hybridization reaction. For
example, a very low level of posteriorly localized olc27
mRNA, which lacks both olc15 and olc16 regions, can be
detected with extended development of the in situ hybridization detection reaction (Fig. 5A).
No striking repeated sequences, which might be expected
for redundant binding sites, were found within the osk
3′UTR. However, RNA-binding proteins frequently
recognize both structural and sequence elements and
multiple copies of a binding site for one such protein might
not be obvious from simple sequence comparisons.
oskar mRNA localization elements
Conservation of localization elements and
localization mechanisms
In Drosophila, a number of maternal mRNAs are known to
be localized during oogenesis. How similar are the mechanisms used to localize these mRNAs? For osk mRNA, we
find that at least some cis-acting elements can act independently of the entire localization signal, suggesting that they
could also be used in partially localizing other mRNAs.
Many localized transcripts, including osk, share a similar
first step in localization, involving specific early movement
into the oocyte from the nurse cells (Whitfield et al., 1989;
Raff et al., 1990; St. Johnston et al., 1991; Golumbeski et
al., 1991; Suter and Steward, 1991; Lantz et al., 1992;
Cheung et al., 1992; Aït-Ahmed et al., 1992). Although there
are differences in the timing of movement into the oocyte,
some of these mRNAs might also use the localization
element identified by olc15 and olc16. Similarly, the osk,
fs(1)K10, and yemanucleinα mRNAs, which are all transiently concentrated at the anterior margin of the oocyte in
a process requiring capu and spir, might share a localization
element. Computer comparisons of the sequences of various
localized mRNAs (see Materials and Methods) do not reveal
any striking homologies. However, the sequences mediating
localization of these other mRNAs are currently at best only
roughly mapped (Cheung et al., 1992), and it is difficult to
ask if cis-acting elements are in fact conserved. Our results
identify regions of approximately 100-200 bases in the osk
3′UTR which contain RNA localization elements; if the
elements themselves are small and/or contain variable
nucleotides at some positions, we will need to know much
more about the specific nature of the putative factor-binding
sites before we can detect them in other RNAs using
sequence comparisons.
In addition to the possible conservation of cis-acting
localization elements, there are also suggestions that basic
mechanisms may be conserved. In particular, hybrid
osk/lacZ transcripts from many of our transgenes transiently
accumulate at apical regions of the nurse cells. As
mentioned previously, these are sites where bcd mRNA
normally accumulates during its movement to the anterior
pole of the oocyte (Stephenson et al., 1988; St. Johnston et
al., 1989). It may be that both osk and bcd mRNAs traffic
through the same location, but that osk normally does so
without interruption while bcd has a programmed pause. As
this step in the localization of bcd mRNA requires microtubules (Pokrywka and Stephenson, 1991), which are themselves concentrated at the apical regions of the nurse cells
(Theurkauf et al., 1992), movement of osk mRNA from the
nurse cells to the oocyte may also rely on microtubules.
Characterization of the cis-acting osk localization signal
has revealed multiple elements that independently direct
steps in the posterior localization of osk mRNA. Mapping
the fine structure of these elements and identifying factors
that bind them may ultimately allow us to elucidate mechanisms underlying not only the movement of osk, but also of
many other localized mRNAs.
We thank Trudi Schüpbach and Christianne Nüsslein-Volhard
for fly stocks, Vince Pirotta for the CaSpeR transformation vector,
and Andrew Leask, Robin Wharton and Joan Wilson for comments
on the manuscript. Supported by a David and Lucile Packard Fel-
177
lowship (P. M. M.) and an NIH training grant #HD07249-11 (P. J.
W.). P. M. M. is a PEW Scholar in the Biomedical Sciences.
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(Accepted 28 May 1993)