Due to Twitter becoming a garbage can website, with prospect of collapsing, I’m copying here my previous conferences live-tweets.
I’ll do it gradually from last to first and I might edit it to look pretty later-on.
Israeli RNA society meeting, 2022
Conferences Live tweets
On my way to
@RNA_ISR
meeting on Jerusalem!
Gonna be really fun
I am going to use #RNA_ISR for live tweeting.
6:55 AM · Oct 26, 2022
Meeting program:
https://docs.google.com/document/u/0/d/1Cl-Zcsuh0A_qVEKXFNf47aM-qcDCsNZ2x3dZmGd-e70/mobilebasic
Here we go
#RNA_ISR
9:38 AM · Oct 26, 2022
Prof. Ruth Sperling talking about Yossi, who passed away 10 yrs ago. The meeting is named in his honor.
We begin with Lynne
@MaquatLab
Giving the first keynote, on NMD
9:50 AM · Oct 26, 2022
NMD is adaptive to environment. Todayshe will talk about the tole of UPF1 & fragile X
UPF1 & Phospho-UPF1 interacts with FMRP. https://www.nature.com/articles/s41556-020-00618-1
Looking at NMD in fmr1 KO mouse
https://ncbi.nlm.nih.gov/pmc/articles/PMC8597091/
Now talking about new data (in press) on omics. Using SILAC showed FMRP binds mRNA, depends on Pabpc Seq shows ot primarily binds tge 5′ region.
*binds & block translation
Nice data looking using reporter on binding, trnbslation, polyA length.
In KO polyA tails are shorter. This protects the mRNA from decay as well.
https://pubmed.ncbi.nlm.nih.gov/36356584/
Next is
@AldemaSasChen
on RNA acetylation & thermostabiliy
#RNA_ISR
10:14 AM · Oct 26, 2022
Nat10 modifies C to ac4C. Only few examples were known. She developed ac4C-seq methods to get whole transcriptome data. In human & yeast found only the known rRNA & tRNA. But in Nat10 o/e see 150 mRNAs. At dpecific motif.
Then mapped in bactetia & archea.
Found in thermotolerant archea in many types.
Nat10 deletion, the archea are now temp sensitive.
https://www.nature.com/articles/s41586-020-2418-2
Dan Bar-Yaacob on mRNA editing in bacteria
#RNA_ISR
10:30 AM · Oct 26, 2022
A-to-I editing was thought to be only in eukaryotes.
Known editing enzyme edits only one tRNA, one site.
Found several edited 15 mRNAs
http://m.genome.cshlp.org/content/early/2017/08/30/gr.222760.117
Now increased to 40 genes.
Creates non-syn codon from tyr to cys
Focus on a toxin encoding mRNA.
Created reporter with edited vs non edited version
he non edited grows fine.
The edited dont grow until some mutants shut down expression.
Very cool and clear new data on the importance of the cys and disulfid bond for toxicity.
New data on editing in many bacterial species.
#newPI
Next is Raphael Benhamou on small molec to target disease associated RNAs
#RNA_ISR
10:46 AM · Oct 26, 2022
#newPI
Almost all drugs are against proteins. All druggable proteins are 0.2% of the genome.
Almost no drugs for RNA, thou many linked to disease.
He describes the approach from @disney_lab for design. Developing RIBOTAC targets ribonuclease to target RNA.
https://www.science.org/doi/10.1126/scitranslmed.abd5991
I was asked to delete the tweet with the pic ause some unpub data there. But unfortunatly deleted entire thread. Heres the link to the paper he described. @BenhamouLab
Back from coffee break, we continue with
@SaharMelamed
on small RNA-mediated regulation in bacteria
#RNA_ISR
11:29 AM · Oct 26, 2022
#newPI
Intro to snall RNAs https://pubmed.ncbi.nlm.nih.gov/32147527/
Usually regulate by base pairing in trans, with hfq, an rna chaperon. Targets can be degraded or translation inhibition
Describes RIL seq to identify targets.
https://nature.com/articles/nprot.2017.115
https://sciencedirect.com/science/article/pii/S1097276516304130
Found 1000s of interactions
Found another protein https://pubmed.ncbi.nlm.nih.gov/31761494/
Mapping in diff bacteria species.
Now looking at the role of sRNAs in community, phages, host interactions.
Poster 40 describes new data on bacterial community
Very cool data showi g diff interaction in solitary culture vs co culture
What happens with phages? Poster 10 and student talk in session 2
In host interaction poster 37
Next is Assaf Bester on lncRNA CYTOR, epigenetics in cancer but will begin on methods to identify lncRNA function
#RNA_ISR
11:48 AM · Oct 26, 2022
Machine learning on crispr screens.
Identifies ~85% hits.
Most seems to inv e cis regulation, but also transcription, enhancers.
For each lncRNA gas a score rekated to function.
Tested 40 lncRNAs, and 50% showed the preficted growth phenotype when knocked down.
CYTOR one of the predicted that requires for growth.
See changes in chromayin organization, replication, epigenetics.
Corrated with poor survival of cancer.
Suggests tgat crispri screen has lits of falsezpos rate because use 1 gRNA. Uding 2 gRNAs together works better.
Next is Isana Veksler-Lubinsky on miRNA in worms.
#RNA_ISR
12:02 PM · Oct 26, 2022
Specifically in miRNA isomiRs
These have either 3′ or 5′ or both changes.
The 3′ has same seed seq. Most common.
5′ has shifted seed
Looking at isomiRs along developmentm
Only about 10% of reads match to isomiRs, 20% in dauer stage.
But for looking at specific, go up, down at specific stages.
Sorry, i got lost. Too many miR names and expression graphs
Last is Boris Slobodin (ex phd from our lab now #newPI)
Will talk about the 5’utr of #SARSCoV2
#RNA_ISR
12:19 PM · Oct 26, 2022
How sars2 repress host genes, yet promote expression of its own genes.
Was know from sars1, that NSP1 is msjor factor affecti g hist mRNA half life
So does nsp1 of sars2 but mostly spliced genes. But many antiviral has but one exon.
Looked at translstion, see reduced polysomes
Nsp1 mainly binds 40S, 80S, so affect initiation.
How does it differentiate host to viral?
Can see when using the 5’utr of tge virus more nsp1 protein.
Found stem loop in 5utr that is important.
Actually the most important is no G close to the cap
Found also that the 5utr confers cap independent tra slation, like IRES.
The subgenomic 5utr is cap dependent.
https://pubmed.ncbi.nlm.nih.gov/35849342/
This was recently pub
Next is keynote @CChengLab
on LINE derived splicing and RBP in immune response
#RNA_ISR
12:37 PM · Oct 26, 2022
Unpub new data so i will not tweet in detail.
Found an RBP that mask cryptic splice sites.
These can cause dsRNA which activate dsRNA sensors and inflamation.
In KD found >1000 cases of such cryptic splicing leading to msny consequences.
These sites are enriched for LINE (using iCLIP).
The structure predicts dsRNA.
These varisnts go out to cyto, by fractionation & smFISH. Pretty pics!
So can be sensed by dsRNA sensors.
Indeed see expression of interferon response genes.
KD of dsRNA sensor alleviates it. Cool.
Cancer!!!
Lunch!
And posters…
#RNA_ISR
1:02 PM · Oct 26, 2022
Next parallel sessions about to begin. Im in session 1, 2nd in line. So i will live tweet only after my talk.
Someone live tweet me please
@RNA_ISR
#RNA_ISR
2:49 PM · Oct 26, 2022
Hadas Biran present a new algorith called SPIRAL.
Nice presentation.
#RNA_ISR
3:30 PM · Oct 26, 2022
Next is Roni Cohen-Fultheim on A-to-I editing & autoimmune
#RNA_ISR
3:32 PM · Oct 26, 2022
From Erez Levanon lab.
Anti viral MDA5-MAV pathway of dsRNA sensing can induce autoimmune.
Endogenius dsRNA are edited so not to be identified.
Goalv to find immunogenig dsRNA tgat are not edited, thus can triger autoimmune
Looking at inverted repeats at 3UTRs, and looking at editing there.
High editing indicates high risk of this dsRNA as immunogenic.
Some key unpub work showing how editing is related to an autoimmune disease in mouse model.
Very cool.
Now Eden Engal from @MaayanSalton
lab on alt splicing in FSHD2 a muscular distrophy disorder.
#RNA_ISR
3:49 PM · Oct 26, 2022
SMCHD1 is the mutated gene.
It is a chromstin binding, involved in gene dilencing, x-inactivation.
SMCHD1 was found as possible regulating splicing.
See alt splicing in patients. But not in the diff expressed genes.
So separate roles.
About 300 alt spliced (30%) can be related to the disease.
Focus on one gene.
A gene in which mut is already linked to the disease.
Found molecular link. Nice
Next is Anna Uzonyi from
@SchragaSchwartz
lab
On m6A modification & splicing
#RNA_ISR
4:01 PM · Oct 26, 2022
https://www.biorxiv.org/content/10.1101/2022.06.29.498130v1 Excellent talk.
Anna won best talk award in our session.
The final, keynote, talk by @HentzeTeam
on riboregulation of metabolism.
#RNA_ISR
4:51 PM · Oct 26, 2022
He is talking about how his lab found 1000 proreins that lack known RBP domains.
And known to have other functions.
Reverse dogma- not protein that regulate RNA but the RNA regulate the protein.
Fir example this really cool paper
https://www.cell.com/cell/fulltext/S0092-8674(19)30093-5 ·
And now descdibing this amazing work
https://www.sciencedirect.com/science/article/pii/S1097276522004865
This new work in animals:
https://www.biorxiv.org/content/10.1101/2022.02.10.479897v1
Excellent talk, very clear and thought provoking.
Had lovely dinner with many of today’s speakers (and other guests).
#RNA_ISR was a great meeting and i already look forward to next year’s meeting (
@WeizmannScience !).
This concludes my RNA conference blitz: 4 meetings in 4 months.
Next meeting will be https://conf.fiseb.org/
8:30 PM · Oct 26, 2022
EMBL The Complex Life of RNA 2022
“The complex life of RNA” meeting
@embl @EMBLEvents
is just two days away! Here’s the program: https://embl.org/about/info/course-and-conference-office/events/ees22-10/… If you are coming to the meeting, come visit my poster number 77 (odd numbers are on day 2). Follow #EESRNA for live tweets.
Had lots of fun meeting with
@Murat__Sunbul
and his RNA group this morning.
Now i’m at @EMBLEvents
#EESRNA meeting, which begins in a few minutes!
2:54 PM · Oct 12, 2022
After nice introduction (& funny safety movie!), we now begin with keynote Elena Conti
@LabConti and she will talk about the RNA exosome.
#EESRNA
3:18 PM · Oct 12, 2022
Many functions in cyto &nuc. Focus on nuclear functions.
10 subunit core + co factors + substrate adaptors.
Co factors are mostly helicases different for nuc & cyto exosome.
Blocks the entry site but then helps the RNA into active site
Such helicase is mtr4.
It binds specific adaptors fir specofic RNAs.
Complexes like NEXT, PAXT, nop53 and more.
So much work done to see the structure of all these complexes
Stil more questions of specificity.
Probably more adaptors which interact with the more general adaptors.
What other on the mRNP signals for degratadation?
Cap, pA tail
Splicing
In yeast: THO & sub2 -> export
Isolating THO-mRNPs -> rna-seq
Enriched for long RNAs.
Doing cryo-ET -> compact particle.
It is highly enriched in Yra1
Yra1 has unstructured tegions RGS rich
Velcro mechanism?
Consistent with in vivo data that compacts the mRNP that is releived when polyA is added.
This can drive processive vs destructive pathways.
Very nice ideas
I wonder if such proteins work to compact transfered mRNAs through nanotubes.
And mRNA multiplexing
Next is olivier duss on transcription-translation coupling (in prokaryotes i guess…)
#EESRNA
Using single molecuele imaging on immobilize dna, rna, polymerase
https://pubmed.ncbi.nlm.nih.gov/31761533/
Now new data on transcription-translation
Reconstituted all components of both complexes. Wow!
Very cool approch to monitor both processes
They can start with diff conditions e.g. coupled, collided, uncoupled, polymerase not acting etc…
Can go up to 5 or 6 colors!
Mix & match ribosomes & polymerasevof different organisms
Also looking at m6A modifications & processing by similar methods.
Havecresults of writing & reading.
See many unproductive binding events.
Cool seeing writing & reading on same sample using both complexes together labeled different colors.
@OlivierDuss
Now is María Gómez on how histone H1 regulates ncRNA turnover and m6A regulation
4:34 PM · Oct 12, 2022
Using H1 TKO ES cells (50% reduction of H1)
Looked at transcription SNS-seq & replication
https://www.nature.com/articles/s41467-018-03539-8
See accumulation of lncRNAs and asRNA at chromatin in H1TKO cells.
Why? Looking for RNA modif, focus on m6A.
Based on prior interactors with H1
Indeed reduced m6A in lncRNA in H1 TKO.
Very nice https://www.cell.com/cell-reports/fulltext/S2211-1247(22)01157-3
Last before the coffee is Maja Gehre postod from Bernnecke lab
On heterochromatin & transcription.
Examples are piRNA clusters in fly
Regulated by Rhino with other fly funny name proteins.
Found i heterochromatin.
It is a chromatin remodeler.
ChIP data shows transcription & Rhino but lacking canninical histone modifications!
No poly A
Using auxin depletion of Rhino showing it required fro elongation but not initiation.
Using turboID to find other associated factors.
Very nice work
And we’re back.
Next is Alexandra Chivu on pol II pausing.
5:45 PM · Oct 12, 2022
@alexachivu
Think this step that is prevalent in metazoa is a form of additional regulation.
Using precision run-on (PRO)-seq 22 organisms from bacteria to human.
Correlats to NELF evolution
Very cool data seeing upon depletion of NELF factors and seeing different clusters
Hox are transcription factors regulate embryonic development.
Found it interacts with splicing factor.
Her work is here
https://academic.oup.com/nar/article/50/2/763/6471869
@CarnesecchiJ
Next is Julie Carnesecchi.
She will talk about Hox and splicing
#EESRNA
6:02 PM · Oct 12, 2022
Next is Manuel Carminati from
@lapassmore
lab on mrna 3’end processing
#EESRNA
6:16 PM · Oct 12, 2022
Do CPF or APT complexes associate w polii directly? How?
Shows using pulldown
Deletion of CTD (after pulldown) tge interaction retained.
Surprise!
CryoEM shows it sits next to RNA exit channel.
When uding ppase subcomplex found polII dimerization!!a 🤯
Dimerization is due to CTD de-p.
https://biorxiv.org/content/10.1101/2022.07.28.501803v1
Cool work!
Jens Bohnev is next, on U1, checkpoints and incorrect polyA
6:32 PM · Oct 12, 2022
How does U1 supresses cryptic pA sites?
There is 1kb window from 5′ splice site.
Some data indicate p of CTD.
Also relates to the inding strength duration of U1 to rna
I got lost how cdk12 and u1 make a checkpoint.
Last for this session is Janosch Hennig on roX ncRNA
G
Unlike in nammals, in fly no x-chromosome inactivation, but x-chr 2fold transcriptionbin males.
Regulated by roX1 &2 ncRNAs.
These rnas are regulated by MLE helicase
He doesnt want to bore us with protein structural details.
Still too many details, man.
Nice bio data showing in vivo function (or at least the localization to x-chr) at the end of talk.
Flash talks!
#EESRNA
7:01 PM · Oct 12, 2022
LncRNAs are translated.
It protects from decay. Rolecfor NMD. Poster 49
I cant follow her talk. Something on nonAUG and ribosome profiling. Poster 91
Ribosome stalling in bacteria and colliding.
A dna repair protein is involved! Cool!
Poster 93
eIF4A in translation initiation.
Using single molecule FRET to study dynamics. Looking at 5′ structure and more
Poster 123
Covid mRNA-1273 vaccine
Dynamics of the mRNA, polyA tail and stability
Poster 127
Unclear. Something about fertility and polyA site
Poster 137
Local translation in axons, m6A and long 3’utr.
Poster 145
mRNA export in yeast, Tho1 & Sub2.
Novel annealing activity of the DNA, R loop resolution.
Poster 151 & 54
Inducible CRISPR ko of RNA exosome subunits.
Poster 159
Last one:
Splicing and 3D chromatin structure.
Poster 163
And now its postering time
Morning session #EESRNA
We begin with Eva Maria Novoa Pardo on the epitranscriptome at single base resolution 🤔
10:00 AM · Oct 13, 2022
Interested in trans-genertional inheritance of RNA in mammmals.
Focus onbhow high fat diet of father affects metabolism of offspring.
Specifically modifications that affect tRNAs to create ingerited tRNA fragments.
Instead of relying on ab or chem to identify modification, uses direct RNA nanopore seq.
Talks abiut advantages & challenges of this method.
One major is nanopore reads rna >200 base.
But most sperm rna is <200.
Also how to identify mod?
https://www.nature.com/articles/s41467-019-11713-9
More
https://www.nature.com/articles/s41587-021-00915-6
Thst work with
@AldemaSasChen
@SchragaSchwartz
Nice unpub data of de novo base calling on rna from yeast, human cells
Multiplexing to be able to use small amount of rna
https://pubmed.ncbi.nlm.nih.gov/32907883/
I missed the paper solving the problem of short non polyA rna seq.
She finally has prelim data on sperm rna.
Cool and good luck!
Next is Nadezda Podvalnaya on the nuclease complex PUCH, piRNA in worm.
10:33 AM · Oct 13, 2022
piRNA is transcribes as a precursor with 5′ cap.
Looking for proteins involved of precursor 5′ processing.
Found two proteins with funny name (yes, worms have funny gene names too).
Got structures, studied interaction and made mutations to study 5′ processing using reporter.
In vitro assay gave nothing.
Searched agsin and found 3rd protein partner.
Now it works!
Shows specificity to the precurser.
Next is
@KristenWLynch1
on alt splicing in human immune system
10:47 AM · Oct 13, 2022
Q not how but if alt splicin contribute t T cel function.
Looking at HDAC7 exon 9 upon t cell activation.
When skipped, the smaller isform is retained in nucleus -> more histone ac -> change in gene expression.
*less histone ac
Back from coffee break (i didnt have coffee, i get my caffein from diet cola).
Now more on RNA modifications with
@TheHornerLab
This time in virus-host interface
#EESRNA
11:47 AM · Oct 13, 2022
Working on the (+) ssRNA HCV
Replicates in cyto and on ER.
Many structures in the genome
Focus on m6A modifications which occur in the nucleus.
How m6A is added if the genome is cyto.
How does it affect the virus?
KO of n6A writer -> more virus in cells.
Found about 20 sites in viral genome.
Focus on 1 validated site.
Mut – No effect on replication but more viral production
Mut -> more interaction with viral core proteins -> more packaging..
What about the problem of how hcv rna is m6A-ed if its in the cyto?
See here:
https://biorxiv.org/content/10.1101/2022.06.27.497872v1
Very cool
Next is Malgorzata Ewa Rogalska on getting splicing networks.
12:17 PM · Oct 13, 2022
Already tmi on the splicing & alt splicing mechanisms.
Moving very fast on the data.
Large scale rna seq to see all alt splice events and creating a map with splicing factors.
I think. Got lost.
At the end showed how her network predict mut in key factirs in tumors based on alt splicing map.
Next is Andres Ramos on non canonical readers of m6A.
12:36 PM · Oct 13, 2022
IMP1 (aka Zbp1, IGF2b1) is m6A reader?
😮
Longer interaction if the zipcode has m6A.
Now he shows a bunchbof NMR maps & structure showing the interaction where m6A sits.
The interaction is different than cannonical YTH domain.
Made mutant that rejects the m6A firm. Plans to thenbfo iCLIP.
Prelim analysis in neurons.
Shows differences in rna, protein and affect neuron dev.
Last for this part is Bertrand Séraphin on m6A reader in yeast meiosis
12:52 PM · Oct 13, 2022
In yeast m6A present only during meiosis (is that true?)
No known eraser and one reader: pho92
Pho92 expressed only in meiosis.
Del pho92 lag and less efficient sporulation.
Even worse in del of writer.
Looked at gene expression during meiosis in the two del strains.
Gene expression dsta correlates nicely with phenotype data.
Flash talks #EESRNA
1:09 PM · Oct 13, 2022
Posterc153 on erk lin28a, rna decay & morphogenesis
Poster 171 dose compensation in fly looking on rbp binding to rna using single molec imaging using TIRF
Poster 183 tRNA, tRNA modifications & influenza A.
Can targeting tRNA modifications have therapeutics opportunity?
Poster 189 m6A Rna stability and X chr & autosome chr dosage compentation
Poster 211 CUT transcripts in yeast rapidly degraded. Connection to polyA polymerase.
Poster 217 neuronal differentiation, transcription & mRNA decay.regulated by PHF3
Last.
No poster number.
5′ splice site remains tethered to pol II
221 based on the program
Lunch!
Back from lunch!
We continue with Stefan Ameres
@AmeresSL
on RNA decay.
Uridylation as antiviral defence in fly
2:44 PM · Oct 13, 2022
U addition by TRUMP copmlex and read by Dis3l2. Exosome subunit.
But extra exosomal function.
U also added to aberrant ncRNAs.
embopress.org
Molecular basis for cytoplasmic RNA surveillance by uridylation‐triggered decay in Drosophila
The identification of an enzyme complex degrading aberrant RNA in flies deepens our understanding of cytoplasmic RNA surveillance and sheds light on the emerging role for RNA modifications.
Poster 202 for kinetic model of U as QC pathway for small ncRNAs.
Looking for new targets post viral infection.
Found novel transcripts that are U.
KO of the U complex, dis3l2 leads to increase in viral capsid.
Found this is same pathway as siRNA (double ko with Ago2)
Looking at siRNA cleavage of reporter see U addition at cleavage site.
If i understand correctly the U addition helps release the siRNA because now the seq (of the cleaved RNA) do not match the siRNA.
Very cool and clever mechanism.
Next is Sara Zaccara on m6A readers and mRNA decay
3:17 PM · Oct 13, 2022
https://pubmed.ncbi.nlm.nih.gov/31520073/
Summary on the 3 YTHDF proteins and their function
All m6A sites can be bound by any of these proteins https://www.sciencedirect.com/science/article/pii/S0092867420305742
Also similar affijity, similar protein interactions, similar localization.
Do they have similar function?
Interesting from this paper when KD one paralog others increase.
They are redundant, and just one function – stability.
Cool. Found small molecule of all 3 paralogs
https://pubs.acs.org/doi/full/10.1021/acsptsci.2c00008
Now doing crispr base editor screen to see which sites im YTHDF2 protein are functional in cell survival (on del 1 &3 background).
Last for this session is Hana Cahova on NAD capping and HIV infection
3:50 PM · Oct 13, 2022
From Jeschke lab herecat Heidelberg. Just met with
@Murat__Sunbul
from same lab yesterday
NAD-capped rna found in prokaryotes h eukaryotes.
In mammals many snRNAs
NAD rna decrease upon hiv infection.
Show NAD-U1 less affinity to hiv rna compared to regulare cap.
Wow she talks fast, cant keep up.
In essence increasing NAD-RNA decreases hiv infection.
And more splicing of cellular rna
Coffee break
With low battery and unstable wifi, we carry on to the RNA trasport/localization session.
We begin with Clemens Plaschka on packaging mRNA in the nucleus.
4:40 PM · Oct 13, 2022
@plaschka_lab
Focus this talk on EJC & TREX complexes that mark and select mature mRNA for export.
In human cells
How TREX select mature mRNA selectively?
Isolate TREX complex or in vitro assemble recombinant subujits.
ALYREF (yra1 in yeast) interacts with EJC-RNA
Next is Mainak Bose on RNA granules in fly
5:16 PM · Oct 13, 2022
Wifi barely works
@mainakB78580463
presents this work
https://cell.com/cell/pdf/S0092-8674(22)00202-1.pdf
Next is Thomas Duchaine of how phosphorilation of decaping enzyme and p bodies
5:32 PM · Oct 13, 2022
Many q about p bodies. Function, redundancy of scaffold, dufferent species, cell types
In worms.
Dcap2 mut is visble but defective in egg laying.
In western blot multiple bands along development. Also diff partners along diff stages
Defects in edc3, 4 affect p granules during embryogenesis.
They also affect interactors of dcp2
Lots of data but out of time and diesnt seem to be close to end
Last is Florence Besse on mRNA granules in neurons
5:48 PM · Oct 13, 2022
Working on Imp (zbp1) in fly
Know to be important for mRNA loc & translation repression.
Found in granules in celll body in fly brain. In most cells not targeted to axons & dendrites, except in mushroom body axons.
Specific targeting of Profilin mRNA (imp target).
Using FISH
I knew this story was familiar https://twitter.com/grnfluoresceblg/status/1549303807858941952
Flash talk! #EESRNA
6:15 PM · Oct 13, 2022
Poster 139 ncRNA raiA in pathogenic bacteria C. difficile
Deletion affects metabolism, toxin h tRNA
Poster 227 localized translation in worm.
Using Suntag successfully.
ERM-1 translated on cell membrane.
Trying to mis-localize translation by tethering looking at physiology
Poster 231 m6A in CD4+ T cell activation.
Decay of CD40L mRNA one example of effect.
Poster 48 trascription start site and mRNA isoforms (by alt splice and slt 3’utr) in fly brain.
The efects works via epigenetic marks
Poster 52 3′ uridylation on rRNA maturation
Poster 60 role of THO complex in pre-export mRNP using TAP purification from yeast and cryoEM
The morning session begins in a few min with
@KarstenWeisLab
on mRNA transport during stress.#EESRNA
He gave a nice tsak on RNA helicases at #EMBORNAloc
How is mRNA directionally exported?
Mex67 is a shuttling receptor that is removed by Dbp5 helicase in the cyto.
Removing Dbp5 by degron causes export block.
But Mex67 amount or loc doesnt change.
Tethering Mex67 to the cyto side of NPC -> export is fine.
To dissect the process, trying to follow mRNA export in live cells by pp7.
Using costum microscope for improved snr.
Awesome movies
Also fast imaging 15ms/frame and de-noising for particle tracking.
Problem that export cases are rare events.
Now with Dbp5 degron, the mRNA accumulate in puncta at nuclear periphery.
Looked at RNP proteins. Some not affected but 1 was affected.
This has condensate properties, that are dissolved by Dbp5.
What is the physiological relevance?
In glucose starvation dbp5 accumulate at NPC and also see the phenotype of that protein with Dbp5 degron.
What is the consequences?
What about export of stress mRNAs?
Export corelates with condensation mutants.
Nice data on diff retention of mRNAs in the mutants thatcrelate to growth conditions.
Very cool.
Next is Patrik Polák on nuclear exosome and QC of mRNA #EESRNA
10:35 AM · Oct 14, 2022
Targeting complexes NEXT & PAXT
These are linked via cap binding protein.
Focus on one of PAXT subunit, mutating looking fir decay functions.
Short acidic motif at N-term required.
Lost interaction to another subunit.
Saw 2 groups of RNAs degraded depends on mRNA length.
For short mRNA look similar to NEXT substrate.
Is there share factor, mechanism?
Actually no. Both complexes compete so if kd the NEXT factor PAXT degrades better.
I misunderstood, not compete. When NEXT is less active -> less use of the factor -> this factor inhibits PAXT.
High NEXT activity -> more use of the factor by NEXT-> less inhibition of PAXT.
Next is
@cosminribo
on de-Adenylation and decay in yeast
10:48 AM · Oct 14, 2022
Negative correlation of polyA tail length and mRNA stability.
Showing mathematical model.
Not sure i understood the model.
Decapping rate correlates to hsllife?
Ccr4 degron see i creaee in tail length but not change in stability
Dcp2 degron stability increase
Back from coffee break with David Gatfield on Smg6, NMD and circadian clock 😮
11:42 AM · Oct 14, 2022
Circ clock regulated by Bmal1:clock >>per:cry
Per protein accumulation measures time.
How regulated?
NMD is surveilance but are there other roles?
Made smg6 conditional mutant at nuclease domain.
See expected effect on nmd substrates.
Looked at curcadian in fibroblasts & liver ex vivo tissue and see changes in clock.
Also in whole animal wt in brain but 27hr clock in liver.
The phase shifts in both regular day night feeding and when feeding not in sync.
At molec level cry2 mRNA levels diff in smg6 mut
Very nice molecular analisys that explains the long period length.
This work is published on biorxiv https://www.biorxiv.org/content/10.1101/2022.07.01.498406v1
Next is Auriane Rakitch on an rna binding protein Mmi1 in S pombe
12:01 PM · Oct 14, 2022
Interacts with Ehr1 which is required for mmi1 binding to rna and chromatin.
Tether mmi1 to mRNA by lambdaN boxB and look at rna decay in light of ehr1 interaction. Nice clear result.
Shows also a role in splicing, intron retention. again ehr1 is required.
Mutation in ehr1 separates the function of rna, chromstin bindung and the role of mmi1 in splicing.
Nice clear presentation.
Now is
@BeAScientist
on ORF and gene expression regulation
12:13 PM · Oct 14, 2022
Syn codons are not equal during translation.
tR A abundance, codon usage etc..
Optimal codons will make more protein. This is know from the 80s.
Why?
We know about transcription, translation etc.. andcrecently nRNA stability.
Nonnoptimal leads to slowed codons -> more deadenylation but also involvment of ccr4not complex.
Where along the cds where codon optimailty is might have diff effect.
Translation ramp know about codons at start of orf.
So in this assay put 1kb tag at begining to avoid ramp problems
Interesting data on opt vs non opt at mRNA vs protein.
Non opt show 2-fold less protein per mRNA.
Conserved human & fly
Why this effect?
Can be misfolding->degradation of the protein or slow translation.
Found no diff in protein stability.
Did single molec translation imahing w
@tim_stasevich
Found fewer ribosomes on the non opt cds.
Very cool.
This is opposite than expected: slow translation should accumulate more ribosomes.
Is there early termination (drop-off)
Or initiation events.
Not detrcting truncated protein product.
Very clever assay to measure dropoff.
And another nice assay looking at deA using MALAT1 3’end instead.
DeA is mot redponsible for the low initiation.
Very cool research. Interesting.
I think this talk invoked the highest number of questions from the audience 😁
Many ppl find it interesting and important great.
Flash talks!
12:51 PM · Oct 14, 2022
Poster 82 mRNA ageing and cap type 2 methylation
Why this hapoens and how this happens?
Interesting.
Poster 96 on dsRNA protein interaction of Sindbis virus which is +ssRNA virus.
Poster 112 on 3’end processing. PolyA vs histone (non polyA) mRNAs.
Structural data.
Poster 120 on viral RNA interactome.
Cytoplasmic virus find nuclear proteins.
Poster 130 role of Smg1 & Smg8 in NMD.
Poster 138 on deadenylation at dingle nucleotide resolution.
Back from lunch with @chekulae
Based on the title, she will present this work from #EMBOrnaloc:
https://twitter.com/grnfluoresceblg/status/1549407100044038146?t=TNCLjuiBHcQyj5XUiPGaYg&s=19
2:27 PM · Oct 14, 2022
Zoom talk because she has covid 😕
Great talk (again).
Get well soon, Marina
Lexi Gao is next, on tRNA expression and cell identity
3:02 PM · Oct 14, 2022
In human 600 genes. Can be 2 dozen per isotype.
Looking at tRNA seq and cell identity.
She talks really fast.
But less diff in isodecoders.
Suggest buffering based by highly expressed isotypes.
Is looking at pol III subunits, doing ChIPseq for pol III during differentiation.
Sees 3 gruops: housekeeping, restricted group and restricted in differentiated cells.
Can see specific prokoter elements to the different groups. Shows activity by crispr editing of these elements.
Next is Lulu Yi-Ju Tseng on QC of ribosomes RAN translation of non AUG initiation
3:17 PM · Oct 14, 2022
CUG repeats or other GC repeats e.g. in ALS cause RAN translation which lead to deleterious peptides
Bad wifi!
Using reporter to study the gc rich repeats to look at translation efficiency. Looking at connection to G4 and looking at helicase that resolves it
Detects ribosome stalling.
Looking at RQC pathway. Key factirs NEMF & listerin.
NEFT adds CAT at C term, then listerin Ub the peptide.
Indeed kd of both lead to increased truncated peptides.
OE of these lead to opposite effect.
Show phenotype of kd in fly and patient derived neurons.
Nice work.
Back from coffee break with Katrin Karbstein on QC during & after ribosome assembly.
4:11 PM · Oct 14, 2022
Prev work in her lab QC in the cytoplasm during assembly.
Now on collision related QC.
ATpAse Rio1 binds ribosomes after 18S nuclease prosseccing and release it. If cleavage is incorrect Rio1 binds poorly.
Looking at miscleavage by inactivating pol I and driving rRNA variants from pol II in yeast.
See in some variants no growth but still normal
More physiological relevant is adding the mut on wt background since most times only some miscleave.
See domi ant neg effect.
Now see problem in loading.
Connect it to collision & RQC pathway.
2nd topic: what hsppens under oxidative stress? Does such damage induce QC?
Focus on RPS26>>has 4 cys that bind zinc.
Under oxi, Zn is release.
Mut of the cys is lethal
Are the oxi ribosomes destroyed or fixed?
In vitro Tsr2 releases oxi Rps26.
Under oxi, Tsr2 moves from free fraction to 80S peak.
Shows release oxi-Rps26 from the ribosome.
Leaving rps26-deficient ribosome.
Repair (in vitro) requires Zn-rps26.
Shows repair in vivo! Nice system.
Thinks its not unique to Rps26.
Shows another system for another RP
Next is Cole Lewis on optimizing therapeutic proteins by optimising 5utr
4:47 PM · Oct 14, 2022
Seeing 1000s fold between different 5utrs.
Screen 33000 5utr for in vitro translation + chx to get just one ribosome.
Then isolating and measuring activity. See 200 fold range
Then see similar w luciferase.
As c content increase translation reduces.
Increasing ccc motif more repression.
One finding is that moderna, pfizer vaccine were in the top!
Looking at length of 5utr see effect if going below 80nt.
Using shorter utr see better translation
Its so nice when such screens work so nice.
Im not jelous at all 😭
From
@GilbertLabRNA
Flash talks! (Final flash session)
5:17 PM · Oct 14, 2022
Poster 158 human disease asdociated rRNA modifications using nanopore seq
Poster 184 DDX6 regulation on stress granules + SG & Pbody segregation & docking
Poster 194
@AldemaSasChen
on RNA modifications in various model organisms, lookimg at co servation, environment, thermostability.
Poster 204 on endogenous Cas9 in Campylobacter.
Found Cas9 also binds endogenous mRNA.
Very interesting.
Poster 220 on the 3utr of mRNA encoding Roquin protein and regulate stability.
Poster 234 on the oposing roles of eIF4A1 & 2 in tumorigenesis.
Poster 246 the role of reversible RNA ADP-ribosylation 😮
This is a novel RNA modification.
The 1st speaker is
@ChaoLabFMI
I wonder if he’ll talk about this work https://twitter.com/grnfluoresceblg/status/1550043718337658880?t=xXtqILNveLCulWBawpWdHg&s=19
Or something new.
10:30 AM · Oct 15, 2022
Cool, unpub work
Jeff is describing all his recent systems fromsimple FISH & MS2, to TREAT (to measure decay), to Suntag with the IRE and TREAT.
Very cool data on the connection between translation & decay.
Now looking on his miRNA work which i discussed here
The first part was mostly about “basal decay”.
What about highly translated by stable mRNAs?
Moving on to talk about 5’TOP motif and LARP1.
*but stable
Nice but puzzling results relating Larp1, eIF4EBPs and 5’TOP
Seeing difference between the reporter to endogenous 5’TOP.
Next is Morgane Corre on alt splicing upon Listeria infection
11:04 AM · Oct 15, 2022
One gene that show switch is CIRBP, which is an RBP.
Is this related to virulence?
Yes. Even the toxin itself induces the switch.
True for other pore forming toxins
Found upstream pathway leafing to this.
See changes in RNA binding of CIRBP and effect on survival. Nice
@Morgane_Corre
Had wifi prob so didnt live tweet Ayala Shiber’s talk. #EESRNA
She showed unpub data looking at co-translational assembly of protein complexes.
Looking at biophysical properties of the subunits and how co-assembly contribute & required for stability
12:10 PM · Oct 15, 2022
She looked at ribosome composition when translating misfolded proreins.
Finally looked at co-localization of mRNAs encoding subunits of same complex.
Sadly, for this last part she didnt mention our work
https://elifesciences.org/articles/66050
Here’s our review on this subject https://www.sciencedirect.com/science/article/pii/S0168952522001974
Next is Edouard Bertrand on single molec imaging & local translation
12:17 PM · Oct 15, 2022
Dual prorein/mRNA localization screen for human 520 genes tested but only 7% localize. Weird.
Looked at ER, endosome etc…
Many loc to p bodies.
Found also translation factories for a few genes.
Cool data on beta-catenin also in intestine tissue
Wow, translation & protein degradation at same site
Very mice looking at mRNA encoding centrosome protein during mitosis.
Very cool Suntag movies!
Spindle! Beautiful
Screened 600 centrosomal encoding mRNAs high throughput.
Only 8 localize to centrosome, spindle different cell cycle steps.
Compre to fly genes. Nice
Next is
@MarkusHopfler
on decay of tubulin mRNA
12:48 PM · Oct 15, 2022
Depolymerization of tubulin leads to fast mRNA decay.
Their lab found TTC5 as important factor
https://pubmed.ncbi.nlm.nih.gov/31727855/
Wow so clear TurboID results to find another factor.
Just one protein pops up
Sorry got caught up with the talk.
Very nice stepwise analysis of these mechanism from the nascent peptide to recruiting ccr4Not complex.
@MarkusHopfler
here’s the paper i mentioned about Xrn1 https://www.nature.com/articles/s41467-022-34417-z
Last one is keynote talk by Bernd Bukau on co-translational assembly of proteins by ribosome profiling
1:04 PM · Oct 15, 2022
Tested in bacteria cis vs trans assembly
https://pubmed.ncbi.nlm.nih.gov/26405228/
Cis setup of the CDS (operon) is important.
Directionality is important translation of the first cds is required fir co assembly with the 2nd
Now talking on Ayala’s work in yeast.
*Work in his lab
https://pubmed.ncbi.nlm.nih.gov/30158700/
Now talking about the role of Hsp70 protein in the coordinated assembly
https://pubmed.ncbi.nlm.nih.gov/31253954/
Co-co assembly, when 2 ribosomes (disome) on same mrna lead to nasent peptide dimerization.
https://pubmed.ncbi.nlm.nih.gov/33384371/
Now unpub work.
Is co-co mediated by slowing/pausing of the leading ribosomes?
Yes, right next to position of onset of co-co assembly.
What causes pausing?
Eg GG or PP di amino acid
Also factors at nascent chain exit site.
Force by the nascent chain itself in the exit channel.
Interesting.
Fin! #EESRNA Thank you very much to the organizers
@WendyGScientist Anne,
@torbenheick and Ramesh It was an excellent meeting!
EMBO RNA localization and local translation
We’re only a few days away from from the RNA localization meeting! My poster is ready, the 1-min talk is… well, I still need to rehearse it. It’s gonna be great! I will use #EMBORNAloc for live tweets. Stay tuned
So, my flight to #EMBORNAloc is delayed already by 2hrs, and hopefully we’ll take off in 20 min
Thankfully, i will still have ample time for my connect flight.
But, not a great start 😕
I am soooo tired.
6:45 AM · Jul 17, 2022
#EMBORNAloc is starting. Dont know if i’ll live tweet today. Too tired.
But the view to the sea is nice
8:14 PM · Jul 17, 2022
The keynote speaker is Ruth Lehmann.
Btw the official tag is #EMBOrnaLocalization but its too long so i will continue with mine. Sorry
Cool! Using suntag to image translation of nanos in fly germ granules
Nice connecting translstion efficiency with Oskar mutants (and other)
Good discussion of her results.
Then there were 10 flash talks (1min each) which was… good but sometimes difficult to follow (at my brain dead state).
Now food!
#EMBOrnaLocalization #EMBORNAloc day 2 begins in 20min. My poster is up. Breakfast being digested.
Good to go.
10:39 AM · Jul 18, 2022
first speaker is Julia Mahamid from
@embl
she will talk about in-cell structural biology
10:57 AM · Jul 18, 2022
Cellular cryo electron tomography #cryoET
On whole cells in intact state. Corralate with fluorescent imaging and FIB to slice the cell
Also organoids & tissues! https://www.biorxiv.org/content/10.1101/2022.04.12.488077v1
Now, she wants to use cryoET of stress granules in HeLa cells.
Cool
Problems are limit of resolution 10 angsrom and how to identify the protein forming structures
Solution: label-free imaging based on known protein-protein interactions.
https://pubmed.ncbi.nlm.nih.gov/32732422/
Whoa! 3.5A resolution!
These were data from bacteria. She says can even go higher resolution!
https://biorxiv.org/content/10.1101/2021.12.18.473270v1.full
Now back to stress granules.
Ok not SG.
so they found mumps virus infection in their cell culture 😁
Here’s the story. Very cool! https://www.biorxiv.org/content/10.1101/2021.07.10.451879v2.full
Next is Daniel Fürth from Upsala university.
Already several min of tech problems 😕
11:43 AM · Jul 18, 2022
Nothing yet
The crowd is getting restless
@BassellLab
talked about round tables
Ok, finally on.
New PI @furthlab
in situ seq for spatial transcriptome analysis.
Creating cDNA in situ for 3UTRs with rolling circle amplification with barcodes. Imaging.
Then lysing the cells and NGS for the batcoded cDNA
Made atlas of fly larva
Some comparison to other seq methods found some biases e.g. bias to translated mRNA.
Next is Florance Rage from CNRS on imaging translation in motor neurons
12:10 PM · Jul 18, 2022
My chair’s back just broke
Focus on SMN proteins.
SMN protein itself moves along axons.
SMN defects affects mRNA transport e.g. beta actin, Anxa2
Using suntag for translation imaging of Anxa2 in iPSC derived motor neurons.
Looking at dynamics of movement & translation.
This is still work in progress
Last before coffee break is @drosoigmm
to talk about imaging tra slation in fly embryos
#EMBORNAloc
12:26 PM · Jul 18, 2022
https://pubmed.ncbi.nlm.nih.gov/33927056/
Looking now at switching UTRs between Twist & Snail to see how it affects local translation & development but mo results yet…
@binwu1 is the next speaker continueing his work on single molecule translation.
1:05 PM · Jul 18, 2022
Begins with cat pics to demonstrate gene expression stochasticity.
Focus on translation in his talk, of course.
Overveiw of transcription bursting studies to introduce the concept.
Need to image at the hours range to capturr translation dynamics
Tethering the mRNA to membrane with dark pp7, and label with red-ms2.
Thus good SNR and track mRNA for hours.
Opposite to transcription, translation is On state (“burst”) most of the time
Using IF on the scfv-gfp to get intensity of single gfp, then calculate how many per mRNA
But the nature of this system is that there could be overlap of bursting for aparent longer burst time. The longer the CDS the more ovetlap is seen
Can calculate initiation, burst & dwell times.
Shows nice data comparing the burst time with various 5UTRs getting to interesting insights on regulation of translation.
greenfluorescentblog 