1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Polar localization of mRNA can be achieved by three different mechanisms: (i) local
stabilization and regulated degradation of mRNA; (ii) local trapping of an RNA that is
diffusing through the cytoplasm; and (iii) active and directed transport of mRNA.
Although there are examples for all three mechanisms, the latter is the most common
mechanism employed. The transported mRNP complexes form larger structures, which
are referred to as RNA transport granules. These granules are transported by motor
proteins along microtubules and/or the actin cytoskeleton to their final destination. In
addition to the association with the cytoskeleton there is also evidence that mRNA
transport and ER trafficking may be coupled (Schmid et al., 2006; Aronov et al., 2007).
Before mRNPs reach their final subcellular destination, translation is usually delayed by
the recruitment of translational repressors (Schoenberger et al., Plant J., 2012).
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 localization and asp transcripts (B), a more diffuse anterior enrichment.
(C–E) Early and late posterior localization patterns. While both osk and orb transcripts
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).
34
Figure 4. Membrane-AssociatedPatterns (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
transcripts localize within cortical polygonal networks ([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 membrane (arrowheads).
35
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 centrosomes (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 chromatin-associated foci during
metaphase (K), which then become telomeric during anaphase (L).
36
(A–F) Stage 4 (B–E), 5 (A) and 9 (F) embryos hybridized with probes for the indicated
apical (A), cell division-associated (B and C), membrane-associated (D), and nuclear
retained (E and F) mRNAs (red signal, left panels) and colabeled 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). Arrowheads in (E) and (F) indicate nuclei showing an accumulation of kuk and cas
mRNAs, respectively.
37
Subcellular localization of mRNA transcripts correlates with subsequent localization of
their protein products. (A) Fluorescence microscopy images of cells expressing MS2-GFP
(green) and transcripts containing or lacking binding sites for MS2-GFP. “6xbs” after a
gene’s name denotes that the MS2 binding sites were introduced immediately next to
this gene. Cells shown in (l) were supplemented with the fluorescent membrane stain
FM4-64. Transcripts were plasmid-encoded, unless otherwise stated. Scale bar, 1 mm.
(C) (a and b) Fluorescence microscopy images of cells expressing MS2-GFP (green) and a
BglF-mCherry fusion protein red), whose transcript contains the 6xbs. (c) An overlay of
the signals. Scale bar, 1 mm. The possibility of false detection due to fluorescence
leakage between the filters was ruled out. The white arrows point at a cell in which the
6xbs-tagged bglF-mCherry transcripts are detected (a) and the BglF mCherry protein is
not (b) (see also the results in fig. S18).
38
Figure 1. Visualization of Reporter mRNAs in Live Cells (A) Schematic describing the
constructs used in this approach. The system is comprised of two components, a
reporter mRNA and a GFP-MS2 fusion protein. The GFP-MS2 was expressed under the
control of the constitutive GPD promoter, while the reporter mRNA was under the
control of the GAL promoter. The reporter mRNA contains six binding sites for the coat
protein of the bacterial phage MS2. To avoid possible interference with translation and
the function of the 3’UTR, the MS2-binding sites were introduced immediately after a
translation termination codon. The 3’UTRs were either from the ASH1 gene, to induce
mRNA localization at the bud tip, or from the ADHII gene, as control. In addition, a
nuclear localization signal (NLS) followed by an HA tag was introduced at the N terminus
of the fusion protein, so that that only the GFP protein that is bound to its target mRNA
would be present in the cytoplasm. (B) Live cells expressing the GFP-MS2 fusion protein
and the lacZMS2-ASH1 reporter mRNA. Arrows indicate some of the particles, usually in
the bud. Bar, 5 mm.
39
40
41
42
43
44
45
46