Dendritically Localized Transcripts Are Sorted into Distinct Ribonucleoprotein Particles That Display Fast Directional Motility along

Dendrites of Hippocampal NeuronsFabian Tubing,* Georgia Vendra,* Martin Mikl, Paolo Macchi, Sabine Thomas, and Michael A. Kiebler

What are the underlying mechanisms for RNA localization?

How can we study these mechanisms in vivo?

How does this relate to the work we do in our lab on the fragile X mental retardation protein (FMRP)?

RNA localization and RNA-binding protein localization go hand-in-hand

After a pioneer round of translation, mRNAs can move into different protein complexes that share constituents.

From: Paul Anderson and Nancy Kedersha, Nature

Reviews, June 2009 From: J. Ross Buchan and Roy Parker, Molecular

Cell, Decemeber 24, 2009

Monitoring RNA localization directly

Some studies look at localization of the proteins, but monitoring RNA localization is also important because there is an assumption that monitoring the RBP tells you about the localization of the RNA when that may not be the case.

Localization of selected RNAs in hippocampal neurons

• Microinjection of fluorescently labeled RNA into hippocampal neurons followed by quick imaging or fixation

• Chose 4 RNAs with known localization patterns; MAP2, CamKII, Histone H3.3, and GAPDH

• MAP2 localized as expected. CamKII did also and they go farther here by removing an element shown to be important for CamKII targeting

1-31nt 31-59nt Rest of UTR

FL1H2H

1H-59nt

CamKIIconstructs

Localization of RNA pairs

• No obvious changes in localization when multiple tagged RNAs are present (RNA isn’t hitching a ride).

• From a technical standpoint, it means you can track multiple RNAs. Also suggests specificity of particles (dendritic versus non-dendritic)

Sept7 and the supplemental dataSeptin 7 (Sept7) was originally identified as one of several RNAs associated with Staufen, an RNA binding protein found in P-body-like neuronal granules.

Tracking RNA particle movement (kinetics)

Saw particles moving towards and away from the cell body at a range of speeds from 0.2 to 1.2m/sec (or faster)

Staufen kinetics to check their assay

• Speeds calculated for CamKII were much higher than previously reported.

• They had already measured Staufen kinetics, so they re-measured using newer equipment and settings.

• Found that Staufen moved faster than what they had previously seen but with speeds that were similar to the RNA transport kinetics.

Back to the Sept7

• Kinetics for Sept7 movement were similar to CamKII

• Lots of immobile particles but some that moved as quickly as CamKII

Back to the Sept7, Part 2

• Since they are seeing similar kinetics, they decide to use dual labeling to measure colocalization of pairs of RNAs.

An in-situ version of figure 5

• Using in-situ with endogenous RNA, they see even less colocalization of Sept7 with CamKII, but still a statistically significant amount.

Their conclusions• Microinjected RNA forms transport-competent

particles that can be sorted into dendrites without passing through the nuclear machinery

• Transcripts are differentially transported into dendrites• Rates suggest motor-dependent transport• Some colocalization of RNAs in particles, but not the

majority of particles, suggesting that specific RNAs form specific particles. Also, no apparent colocalization during transport, only out in the dendrite.

FMRP background information• RNA binding protein that regulates translation• Contains 3 RNA binding domains (KH1,KH2 and RGG

box)• Binds a large number of target RNAs, many of which

contain putative G-quadruplexes• Dendritic localization pattern upon stimulation• RGG box is required for RNP formation and proper

localization• RGG box is required for proper polysome association• RGG box is methylated at four arginine residues

Group participation begins here!

An in vitro method for measuring RNA association via the RGG box

SC1ggc ugc ggu gug gaa gga gug gcu ggg uug cgc agc u

SC1mutggc ugc ggu gug gaa CCa gug gcu ggg uug cgc agc u

Determine amount of FMRP bound

Biotinylated RNAwith G-quartet structure

Fmrp

In vitro synthesized FMRPGG

GG

GGGG

Fmrp

Allow FMRP to bind target RNA and capture complex on beads

GGGG

GGGG

Arginines 533 and 538 are primarily required for sc1 RNA association

Captured protein

Input

SC1 SC1 SC1 SC1 SC1SC1 mut

SC1 mut

SC1 mut

SC1 mut

SC1 mut

533, 538, 543, 545 ∆RGG

533,

538

, 543

, 545

∆RG

G

Arginines

present/ substituted

533, 538, 543, 545

533, 538, 543, 545

533, 538, 543, 545

533,

538

, 543

, 545

533,

538

, 543

, 545

15% 23% 44%percent of WT

0

0.2

0.4

0.6

0.8

1

533, 538, 543, 545

∆RGG

*

*

*

533, 538, 543, 545

533, 538, 543, 545

533, 538, 543, 545

533,

538

, 543

, 545

Blackwell et al., Hum. Mol. Genet., Advance Access published on January 11, 2010

Captured protein

Input

∆RG

G

21 67percent of WT 108 54

0

0.2

0.4

0.6

0.8

1

1.2

1.4

∆RGG

n.s.

**

****

n.s.

**53

3, 5

38, 5

43, 5

45

533,

538

, 543

, 545

533,

538

, 543

, 545

533,

538

, 543

, 545

533, 538, 543, 545

533, 538, 543, 545

533, 538, 543, 545

533, 538, 543, 545

Arginines 533 and 538 are NOT required for AATYK RNA association

Arginines

present/ substituted

Blackwell et al., Hum. Mol. Genet., Advance Access published on January 11, 2010

Our hypothesisMethylation of the RGG box regulates RNA binding and different RNAs bind FMRP differently based on their secondary structure (sc1 with a defined G-quadruplex versus AATYK with a G-rich region).

What we are looking for now is a functional assay for measuring RNA binding in the absence of FMRP or in the presence of different FMRP isoforms (full length, deletion or substitution mutants, etc.).

FMRP

FMRP

FMRP

FMRP

FMRP

A functional assay for monitoring FMRP RGG-box dependent RNA localization (?)

If the sc1 G-quadruplex is a “tag” for FMRP binding, then adding it to an RNA that does not move out into dendrites should result in dendritic localization that is FMRP RGG-box-dependent.

GGGG

GGGG

GAPDH

Mfold, what is it good for?

We can fold short sequences easily and predictably, but not long sequences (anything greater than a few hundred bases)

MS2

GAPDH

sc1LacZ

SV40

sc1

QuestionsWhy does Mfold not allow the G-quadruplex to fold?Why are the 5’ and 3’ ends always in close proximity?What is the upper limit for trustworthy Mfold folding?

What makes up a granule; multiple copies of the same RNA as this paper suggests or 1-2 copies of different RNAs?

Why are so few particles motile? Is this something that is specific to polarized cells?

Are there other functional assays for measuring RNA association in cells?