>> WELCOME TO TODAY'S WALS

LECTURE.

PLEASE TAKE YOUR SEATS.

WE TRYING TO KEEP OUR TIME

BECAUSE THERE IS A LECTURE AFTER

THIS LECTURE.

IT IS A GREAT HONOR TO INTRODUCE

ON BEHALF OF THE NIH FELLOWS

TODAY'S SPEAKER, WITOLD

FILIPOWICZ.

WITOLD FILIPOWICZ COMES FROM

SWITZERLAND WHERE HE HAS BEEN A

GROUP LEADER AT THE FREDRICK

INSTITUTE FOR BIOMEDICAL

RESEARCH FOR THE PAST 28 YEARS.

AND THE PROFESSOR AT THE

UNIVERSITY

UNIVERSITY SINCE 1997.

HE WAS BORN IN POLAND AND

GRADUATED FROM THE MEDICAL

UNIVERSITY.

BEFORE RECEIVING HIS Ph.D.

FROM THE INSTITUTE OF BIOMEDICAL

AND BIOPHYSICAL BIOPHYSICS AT

THE POLISH ACADEMY OF SCIENCE.

WITH HIS Ph.D. STUDIES, ABOUT

TRANSLATION OF RNA PHASE F2, HE

LAUNCHED A VERY SUCCESSFUL

CAREER AND TURNED THROUGH THE

ENTIRE UNIVERSE OF RNA.

HE BROUGHT HIM FIRST AS A

POSTDOCTORAL FELLOW TO THE

UNITED STATES WHERE HE JOINED

THE LAB OF NOBEL LAUREATES AT

THE NEW YORK UNIVERSITY.

HE MOVED TO THE RUSH AN CITY OF

MOLECULAR BIOLOGY IN NEW JERSEY

WHERE HE STARTED WORKING WITH

ALLEN WITH WHOM HE KEPT A

PRODUCTIVE COLLABORATION OF

RETURNING TO THE POLISH ACADEMY

OF SCIENCE AND ESTABLISHING HIS

INDEPENDENT RESEARCH PROGRAM.

HE WAS THEN RECRUITED TO THE

INSTITUTE IN BOS UL BUT KEPT

INITIALLY HIS LAB IN WARSAW.

HOWEVER, WORKING BEHIND AND

TRAVELING ACROSS THE IRON

CURTAIN, BECAME MORE AND MORE

DIFFICULT AND HE ULTIMATELY

MOVED WITH HIS FAMILY TO BOS UL

IN 1984.

DURING HIS OUTSTANDING CAREER,

DR. WITOLD FILIPOWICZ MADE

SEMINOLE CONTRIBUTIONS TO MANY

ASPECTS OF RNA BIOLOGY,

INCLUDING RNA PROCESSING,

ENZYMES REGULATING RNA

METABOLISM, THE ROLE OF

NONCODING RNA IN GENE EXPRESS HAPPEN

TO VISIT THIS COUNTRY AND I'M

QUITE IMPRESSED WITH THE PEOPLE

HERE.

OF COURSE I KNOW MANY PEOPLE AND

I'M GRATEFUL THAT I COULD TALK

TO THEM TODAY.

I TOLD THEM TO COME THEMSELVES.

SO, AS STEP MENTIONED, I WILL BE

TALKING ABOUT THE KINDS OF

CONTROLLING FUNCTIONS OF

METABOLISM OF MICRORNAs AND

METAZEROA.

I WILL SPEND HALF OF MY TALK ON

A FUNCTION AND MECHANISM OF

MICRORNA IMPRESSION AND THEN

WE'LL GO INTO REGULATION OF

METABOLISM OF MICRORNAs.

AND JUST TO START, I WOULD LIKE

TO REMIND EVERYBODY THAT WE ARE

GOING THROUGH SOME KIND OF A

PARADIGM CHANGE IN GENE

EXPRESSION STUDIES.

AND UNTIL RECENTLY, WE KNEW THAT

ABOUT ONE OF 2% OF THE HUMAN

GENOME ENCODES PROTEINS AND IT

WAS NOT CLEAR WHAT THE REST IS

DOING.

AND IT IS NOW CLEAR THAT 90% OF

HUMAN GENOME IS TRANSCRIBED BUT

THIS SMALL PERCENT ENCODES

PROTEINS BUT THE REST ENCODES

NON-CODING RNAs AND WE ARE

TRYING TO FIND OUT WHAT THOSE

NON-CODING RNAs DO AND THIS

SITUATION WAS COMPLETELY

DIFFERENT FROM ECOLEY WHERE 90%

OF THE GENOME ENCODES THE

PROTEINS.

THAT PROBABLY EXPLAINS THE

DIFFERENCE BETWEEN E.COLI AND

US.

SO THERE ARE MANY CLASSES OF EN

NON-CODING RNs  --

RNAs THEY LINK RNAs AND

IMPRINTED RNAs.

A COUPLE OF THEM ARE KNOWN AND

NOW THERE ARE THOUSANDS.

THERE ARE ALSO INTERMEDIATE

LENGTHS EXEMPLIFIED BY SMALL

NUCLEAR RNAs.

WE USED TO STUDY THOSE FOR MANY

YEARS.

BUT THEN THERE IS THIS NUMBER OF

SMALL RNAs, 20 TO HURTY IN

LENGTH AND I WILL FOCUS ON ONE

CLASS ON MICROAND MICRORNAs.

MICRORNAs ARE ABOUT 20

NUCLEOTIDES LONG POST

TRANSCRIPTIONAL REGULATORS.

THEY ARE ENCODED IN A GENOME,

THEY ARE TRANSCRIBED AS LONG

PRECURSORS WHICH FOLD TO HAIR

PINS WHICH ARE REFERRED TO AS

PRIMARY MICRORNAs AND THOSE

ARE PROCESSED IN NUCLEUS BY

COMPLEX OF DOSHA IN THE RNA FREE

ENZYMES INTO SHORTER HAIR PINS

TAKING PLACE HERE, REFERRED TO

AS PREMICRORNAs PROCESSED BY

DICER IN THE CYTOPLASM AND ONE

OF THE STRENGTHS OF THE DUPLEX

IS INCORPORATED INTO MICRORNA

COMPLEX AND ALSO KNOWN AS

MICRORNA RISK TO PROTEINS AS A

KEY COMPONENT.

AND THIS COMPLEXES TO THE 3

PRIME RNA AND IN TRANSLATIONAL

REPRESSION.

AND THIS CLASS OF SMALL RNAs

IS AN ANALOGY ABOUT 1000 OR EVEN

1,500 MICRORNAs MAY OPERATE IN

HUMANS.

MOST OF THE GENES ARE REGULATED

BY MICRORNAs AND THEY

PRACTICALLY REGULATE ANY PROCESS

BEFORE WE ARE LOOKING AT.

JUST TO GIVE YOU SOME PRINCIPLES

HOW MICRORNAs REGULATE MR-NA

TARGETS, THEIR SIDES ARE

STRETCHED THROUGH KILO BASES OF

3 PRIME UTRs.

THEY ARE OPEN LONGER THAN

ENCODING SEQUENCES.

AND THEIR SIDES ARE ENRICHED

NEAR THE TERMINATION CODE

PRESENT IN A ENRICHED REGIONS

AND THE KEY ELEMENTS IMPORTANT

FOR RECOGNITION OF MICRORNAs

BUT THERE ARE EXCEPTIONS TO

THAT.

IS THE SO-CALLED C SEQUENCE

WHICH IS A POSITION NUCLEOTIDE

POSITION 228 IN A MY CROW RNA.

THIS MEANS THE PERFECT BASE

PAIRING TO IDENTIFY TARGETS.

AND GENERALLY MULTIPLE SIDES ARE

REQUIRED FOR A STRONG REPRESSION

AND SOME KIND OF COOPERATE

ACTIVITY BETWEEN MICRORNAs.

WE UNDERSTAND ONE MICRORNA CAN

CONTROL HUNDREDS OF mRNAs

AND ONE mRNA CAN BE TARGET OF

MANY MICRORNAs AT THE SAME

TIME.

SO THERE IS A LOT OF COMPTORIAL

REGULATION.

SO THESE PROBLEMS, MICRORNAs

VERY SUITABLE AGENTS FOR

NETWORKING FOR GENE EXPRESSION

BECAUSE THEY CAN TARGET SEVERAL

mRNAs IN THE SAME PATHWAY,

FOR INSTANCE, AND USUALLY THEIR

EFFECTS ARE SMALL, TWO TO

THREEFOLD BUT SINCE THEIR

MESSAGE CAN BE CONTROLLED BY

MANY MICRORNAs SOMETIMES

STRONGER.

SO THEY SWITCH TO DEVELOPMENTAL

PROGRAMS BECAUSE THIS REGULATORS

ACTING AS RNAs.

THEY DON'T HAVE TO GO TO

TRANSCRIPTION PRO ASSESSING

TRANSLATION.

THEY CAN BE MADE AVAILABLE

FASTER THAN PROTEIN REGULATORS.

BUT THERE ARE ALSO EXAMPLES OF

MICRORNAs FOR OFF SWITCHES OF

GENE EXPRESSION AND I WILL SHOW

YOU ONE EXAMPLE OF THIS SORT

LATER.

SO THIS IS MICRORNA FIELD A

RELATIVELY YOUNG FIELD.

IN 2001, A SECOND RNA TURNED OUT

TO BE C ELEGANS IN HUMANS WAS

IDENTIFIED.

SO THERE ARE MANY, MANY

CHALLENGES AND WE ACTUALLY HAVE

A LOT OF WORK STILL TO DO TO

IDENTIFY mRNA TARGETS AND

MICRORNA FUNCTION IN HEALTH AND

DISEASE.

AGAIN, THIS PRODUCTION OF

TARGETS BECAUSE OF THIS MINIMAL

COMPLIMENTIARITY IS NOT AN EASY

TASKS PREDICTIONS CAN BE ALWAYS

SUPPLEMENTED BY EXPERIMENTAL

EVIDENCE.

SO, WE ALSO START NOW TO

UNDERSTAND MICRORNA FUNCTION AND

TISSUE LABELS BECAUSE MOST OF

THE EXPERIMENTS ARE DONE IN

TISSUE CULTURE BUT NOW PEOPLE

STARTED TO DO KNOCKOUTS AND

IDENTIFY REALLY TRUE PHENOTYPES

OF MICRORNA POSITION.

WHAT I'M GOING TO ADDRESS THESE

TWO POINTS, THE KINDS OF

INHIBITION OF PROTEIN SYNTHESIS

AND REGULATION OF MICRORNA

BIOGENESIS AND THE IMPRESSION

ITSELF AND mRNA TURNOVER.

SO LET ME START WITH MECHANISM

OF INHIBITION OF PROTEIN

SYNTHESIS.

WE AND OTHERS STUDIED THIS SINCE

ABOUT 10 YEARS AGO AND WE STILL

DON'T REALLY EXACTLY UNDERSTAND

HOW THE MICRORNAs THINK ABOUT

THE INHIBITION.

SO, CERTAINLY THE THEY INHIBIT

TRANSLATION AND mRNAs BUT AS

TO THE TRANSLATION OF

INHIBITION, IT IS NOT REALLY

MOST OF THE EVIDENCE INDICATES

THAT MICRORNAs REPRESS

INITIATION IN EARLY STEP BUT

THERE ARE ALSO SOME DATA ARGUING

THAT THEY COULD ACT AS

INHIBITORS.

WHAT IS CLEARLY OR WHAT IS CLEAR

THAT MOST OF THE mRNAs WHICH

ARE SUBJECT TO THE REPRESSION,

THEY ARE ALSO PARTIALLY STRONG

HERE OR LESS STRONG AND THE

DEADD ENALATION IS DEGRADED.

SO WE ARE WORKING ON THE KINDS

OF TRANSLATION INHIBITION OVER

THE YEARS AND OUR DATA SUPPORTS

INHIBITION OF THE TRANSLATION

STEP IN THE EARLY INITIATION

STEP.

FINDING THAT TRANSLATION FROM

IRIS INTERNAL INITIATION SET IS

NOT EFFECTIVE AND ALSO IN THE

WORK DONE IN PARTIAL

COLLABORATION WITH ANOTHER LAB,

WE COULD SHOW THAT IN AN

EXTRACT, THE REPRESSION IS CUT

DEPENDENT.

BUT STILL THERE ARE MANY ISSUES

WHICH WE MAY RESOLVE.

RELATED CONTRIBUTION OF

TRANSLATIONAL INHIBITION VERSUS

mRNA DEGRADATION.

WHY SOME mRNAs ARE NOT

DEGRADED AT ALL BUT OTHERS ARE

STRONGLY EFFECTED.

THE DEADDALATION AND TRANSLATION

REPRESSION INDEPENDENT OF EACH

OTHER OR THE DEADDALATION IS

CONSEQUENCE OF TRANSLATION

REPRESSION HAPPENING FIRST?

AND WHAT FACTORS ARE -- AND HOW

DO THEY EFFECT BOTH PROCESSES.

SO I NEED TO INTRODUCE TWO MAIN

PLAYERS IN A PATHWAY.

THOSE ARE PROTEINS OF A

...

...

AND IN TERMINAL PART, IT'S

ANOTHER REGION AND WHAT THE

STARS INDICATE HERE AND HERE ARE

REPEATS OF TRIPTOPHANES FOLLOWED

OR PROCEEDED FOR GLYCIN, SEAR 19

OR THREAMINE.

THESE 3 REEGINS ARE KEY FOR

REPRESSION AND CCR4-NOT BINDING.

THESE REGIONS ARE DISORDER SO

SEQUENCE PREDICTION AND SOME NMR

STUDIES IN COLLABORATION, FIND

THERE IS NO STRUCTURE IN THIS

REGION.

SO THEY ARE BOTH DISORDERS.

AND THIS ELEMENT, TRANSCRIPTA

FEIGN, RICH ELEMENT, THEY ARE

REDUNDANT SO FOR INSTANCE WE CAN

DUPLICATE IN C TERM SEQUENCE AND

HAVE THE EFFECT OF THE WHOLE

REGION.

SO I WANT TO SHOW YOU ONE

EXAMPLE OF THE EXPERIMENT OF HOW

YOU PROVE THAT TRYPTOPHANS,

THROUGH THIS NONSTRUCTURED

REGIONS ARE CONTRIBUTING TO THE

REPRESSION.

SO WHEN WE TAKE A WILDTYPE

SEQUENCE, AND TETHER IT TO

MRNA, WE SEE ABOUT 10 FOLD

INHIBITION OF PROTEIN THING SIS.

SO FROM THIS LEVEL TO THAT

LEVEL.

IF WE MUTATE SINGLE TRYPTOPHANS,

ANY OF THOSE, WE DON'T SEE AN

EFFECT.

BUT WHEN WE START TO INTRODUCE

INCREASING AMOUNTS OF MUTATIONS,

OF TRYPTOPHANS, THIS PLUS THIS

PLUS THIS, SO WE SEE SOME

EFFECT, 3 MORE EFFECT AND IF WE

MUTATE 7 OR 8, WE COMPLETELY

ELIMINATE THE REPRESSION.

AND WE ALSO ELIMINATE EN

TRANSPORTATION WITH CCR4-NOT

COMPLEX.

THOSE ARE JUST EXAMPLES OF TWO

SUB UNITS THE COMPLEX, CAP ONE

AND C NOT 1.

WHEN WE HAVE WILDTYPE SEQUENCE,

THESE TWO PROTEINS ARE PULLED

DOWN.

WHEN WE TAKE AWAY TRYPTOPHAN, EN

TRANSPORTATION IS GONE.

AND THE SAME APPLIES TO THE N

AND D DOMAIN WHEN WE TAKE THE

ONE WHICH I WILL SHOW ON SLIDES

COMING FROM THE END TERMINIS, WE

FIND THE SAME EFFECT.

TRYPTOPHANS ARE NEEDED FOR BOTH

REPRESSION AND CCR4-NOT BINDING.

WHAT IS MOST IMPORTANT THAT THIS

MOTIF ALSO FUNCTION IN A CONTEXT

OF FULL BW182 PROTEINS AND

MICRORNA REPRESSION BECAUSE MANY

EXPERIMENTS WHICH I WAS SHOWING

SO FAR, THEY BOTH FRAGMENTS OR

MUTANTS OF THE PROTEIN TO THE

mRNA BUT BY DOING DIFFERENT

GENETIC TRICKS, WE CAN NAIL DOWN

THE PROTEINS AND INTRODUCE

MUTANTS TO THE CELL AND SHOW

THAT ACTIVITY DEPENDS IF THEY

REINTRODUCE PROTEINS AND DEPENDS

ON THIS TRYPTOPHAN MOTIF.

OF COURSE, WE WERE VERY MUCH

WORRIED BY THIS EXPERIMENT THAT

WHEN WE MUTATE 7 OR 8

TRYPTOPHANS, EVEN IN A

NONINSTRUCT ORD REGIONS, YOU CAN

MESS UP THIS PROTEIN

FRAGMENT -- WE WERE INTERESTED

IN DOING A GAIN OF FUNCTION

EXPERIMENTS TO SHOW TRYPTOPHANS

ARE REALLY NOT WORKING BY

DISTURBING THE SECONDARY

STRUCTURE OR FOLDING OF THE

PROTEIN.

BUT THAT THEY GENUINELY PLAY A

ROLE IN THE REPRESSION.

SO WHAT WE DID, WE DECIDED TO

TEST WHERE THERE WERE ANY

DISORDER PROTEINS.

FOR ME, THIS WAS AN EXAMPLE.

WILL GAIN REPRESSIVE POTENTIAL,

UPPER INSERTION OF TRYPTOPHAN

RESIDUES NEXT TO GLYCIN, SEA 19

OR 3A MEAN.

SO HERE IS A FRAGMENT OF THE

PROTEIN.

SIC1 IS NONSTRUCTURED SO THE

STRUCTURE STUDIES PERFORMANCE IN

THIS PROTEIN AND WHAT WE DID

HERE, WE JUST INSERTED SHOWN IN

RED, A COUPLE OF TRYPTOPHANS

INTO THE SEQUENCE.

THAT WAS INSERTED.

THEY INTRODUCED MUTATIONS

CREATING GW OR TW REPEATS IN A

PROTEIN.

AND YOU REALLY GAIN THE

REPRESSIVE POTENTIAL.

SO WHEN WE INSERT 4, WE SEE

PARTIAL IMPRESSION.

WHEN WE INSERT 7, WE HAVE MORE

REPRESSION.

AND WHAT IS EVEN MORE IMPORTANT,

WHEN WE INSERT TRYPTOPHANS, THIS

PROTEIN, THE CONTROL PROTEIN,

DOESN'T PULL DOWN WITH THE

COMPLEX, BUT THE PROTEIN WHICH

HAS TRYPTOPHAN IS INSERTED

STARTS TO PULL DOWN THE CODING.

THE CCR4 COMPLEX.

SO THIS WAS VERY IMPORTANT

EXPERIMENT FOR US.

WE ACTUALLY REPEATED THIS WITH

AN INDEPENDENT EAST FRAGMENT

WHICH HAS NO NONSTRUCTURED

FRAGMENT AND GOT THE SAME

RESULT.

SO THIS IS TRYPTOPHAN ROLE IS

NOT REALLY DUE TO THE DISRUPTION

OF THE STRUCTURE OF THE GW182

FRAGMENT.

SO, THIS RECRUITMENT OF THE

COMPLEX CONTAINING DNA SUBUNITS

EXPLAIN NICELY WHY THE

MICRORNAs AND GWRNAs ARE

THEN QUESTIONED BECAUSE THOSE

ARE ACTIVE SUBUNITS SO THIS

WOULD EXPLAIN WHY THE PROTEIN IS

DEACETYLATED.

THE COMPLEX ALSO INTRODUCED

TRANSLATIONAL REPRESSION OF

MRNAs.

AND TO STUDY THIS, WE TURN OUT

TO THE MESSAGES, WHICH ARE DO

NOT CONTAIN POLYA SO THEY

CONTAIN HISTONES AT THE 3 PRIME

END OR THE RIBOSAME, WHICH CUTS

THE MESSAGE HEARSAY AND THIS

MESSAGES THEN CAN BE STARTED

USING THE TETHERING SYSTEM WHICH

I WAS REFERRING TO.

SO WE CAN TETHER THE PROTEINS

WHICH ARE EXPRESSED AS FUSION

WITH A PEPTIDE WHICH TEATHERS

THE PROTEIN TO THE HAIR PINS

INSERTED INTO THE 3 PRIME ETR SO

WE CAN TETHER GW182 OR C NOT

SUBUNITS OR MUTANTS AND CHECK

THE EFFECT ON ACTIVITY.

AND WHAT WE FOUND IS THAT

TETHERING OF THE GW182CEV, THIS

REPRESSIVE FRAGMENT, BUT NOT OF

THE MUTANTS, CONTAINS MUTATIONS

IN TRYPTOPHAN RESIDUES, INHIBITS

TRANSLATION OF mRNA WITHOUT

INTRODUCING ANY DEGRADATION.

EVEN MORE WE COULD DEMONSTRATE

THAT WHEN WE TETHER THE

COMPONENTS OF THE CCR4 NOT

COMPLEX, CAP 1 OR C NOT 1, WE

ALSO INHIBIT ACTIVITY OF

POLYBINDING mRNAs.

IT WAS A HISTONE OF THE

RIBOSAME.

AND THIS REPRESSION IS NOT AS

ASSOCIATED WITH DEGRADATION OF

mRNA.

AS SHOWN HERE BY THE ORDER.

SO, WHAT WE COULD CONCLUDE FROM

THAT, ALSO THIS PROTEIN INDUCED

GENUINELY TRANSLATIONAL

REPRESSION OF mRNAs, THOSE

ARE NOT THE ONLY EXPAND WHICH

SUPPORTS THIS CONCLUSION, I HAVE

NO TIME TO SHOW OTHERS.

WE ALSO CARRIED OUT SOME KIND OF

EP STATIC ANALYSIS TO SHOW THAT

ACTUALLY GW182 PROTEIN FUNCTIONS

UPSTREAM OF CCR4 NOT.

SO WE COULD SHOW THAT REPRESSION

BY GW182 FRAGMENTS DEPENDS ON

CCR4 COMPLEX BUT REPRESSION BY

CCR4 NOT COMPLEX COMPONENTS IS

GW 182 DEPENDENT.

AND THIS REPRESSION WILL STILL

BE TAKING PLACE IF WE KNOCKIT

DOWN.

THIS CLEARLY PITS CCR4-NOT

COMPLEX DOWNSTREAM OF GW182 AND

GW182 IN TURN ATTRACTS CCR4 NOT

COMPLEX WHICH FUNCTIONS AS A

REPRESSOR.

SO, JUST TO SUMMARIZE THIS PART,

THE WGST MOTIFS REFER TO THEM AS

W MOTIFS, TRYPTOPHAN MOTIFS THIS

THIS PROTEIN REPRESENT KEY

ELEMENTS IN INTRODUCING MICRORNA

MEDIATED REPRESSION.

AND THERE ARE TWO CLASSES OF

THESE MOTIFS IN THIS PROTEINS.

ONE CLASS I WAS SHOWING THIS

ELEMENT GW ELEMENTS AND VERY END

TERMINUS OF THE PROTEIN.

THEY ATTRACT ARGONAUT PRO

DISPENSE THEY INTERACT WITH

THOSE PROTEINS BUT THEN THERE IS

ANOTHER CLASS WHICH INTERACTS

WITH CCR4 NOT SUBUNITS.

WE DON'T KNOW HOW THOSE MOTIFS

REALLY WORK IN CCR4-NOT

RECRUITMENT.

HOW IS THIS ADDED TO PLAIN?

THE MOST LIKELY POSSIBILITY IS

THAT THERE IS A SERIES OF WEAK

INTERACTION BETWEEN THE

TRYPTOPHANS AND MAYBE

HYDROPHOBIC MOTIFS IN CCR4-NOT

SUBUNITS.

WE KNOW THAT THIS IS C NOT 1

SUBUNITS WHICH INTERACTS WITH

THE PROTEIN.

BECAUSE IT'S LIKELY THIS

RECRUITS SEVERAL COMPLEXES BUZZ

THEY IR QUITE BIG.

IT WOULD BE REALLY DIFFICULT TO

ACCOMMODATE ON A SHORT PIECE OF

THE PROTEIN.

SO THERE IS SOME KIND OF ANALOGY

HERE MAYBE WITH NUCLEO

IMPORTING -- NUCLEO PORE IN ARE

BASED ON ALANIN GLYCIN REPEATS

WHICH WORKING IDENTICAL ADDITIVE

FASHION AS GW182CCR4

INTERACTIONS.

HOW THE COMPLEX REPRESSES

TRANSLATION, WE DON'T KNOW.

SO BEFORE I MOVE TO THE FINAL

CONCLUSION OF THIS PART, I JUST

WANTED TO MENTION ANOTHER

IMPORTANT OFFERING, ANOTHER

IMPORTANT QUESTION.

WHAT IS REGULATING CREATIVE

CONTRIBUTION OF TRANSLATIONAL

INHIBITION VERSUS mRNA

DEGRADATION?

ARE THE TRANSLATION REPRESSION

INDEPENDENT OF EACH OTHER?

OR THE DEADENYLATION IS THE

CONSEQUENCE OF TRANSLATION

INHIBITION?

AND THERE WERE ALREADY SOME

INDICATIONS IN THE LITERATURE.

THIS ACTUALLY AN EXPERIMENT DONE

IN A LAB WHO IS A COLLABORATOR

OF OURS, WHICH DEMONSTRATED

INVITE ROW,

TRANSLATIONAL -- IN-VITRO,

TRANSLATIONAL REPRESSION COMES

FIRST BEFORE THE DEADENYLATION.

SO MAXIMAL REPRESSION IS

ACHIEVED IN ONE HOUR AND IF ONE

LOOKS AT DEADENYLATION,

DEADENYLATION IS IN THIS

LITERATURE.

WE DECIDED TO LOOK AT THIS IN

HUMAN CELL LINES BY CONSTRUCTING

INDUCIBLE REPORTERS AND PUTTING

THEM INTO SPECIFIC LOCUS IN A

HELA GENOME AND UNDER CONTROL OF

INDUCIBLE REPRESSOR.

SO WE INTRODUCED INTO THE SAME

LOCUS EITHER CONTROL REPORTER,

FIRE FLY LUCIFERASE OR A REPORT

CHER HAS THE 3 PRIME UTR AND

ESTABLISHED TARGETS OF

MICRORNAs OR THE MUTATED

VERSION OF IT.

AND WE LOOK AT DIFFERENT TARGETS

CONTAINING MULTIPLE SIDES.

CONTROL FIRE FLY RECEPTORS, THEY

ARE EXPRESSED IN THE SAME WAY,

INDEPENDENT IF LUCIFERASE

CONTAINED THE 3 PRIME UTR.

BUT WHEN WE HAVE THE WILD 3

PRIME UTR.

IT IS STRONGLY REPRESSIVE.

HERE WE COMPARE A MUTANT ROUNDED

UP WHICH IS NOT REPRESSED BY

MICRORNAs.

IT IS REPRESSED AND WITH MANY

CONTROLS TO SHOW THAT THIS IS

MICRORNA REPRESSION AND THEN WE

LOOK AT THE KINETICS AFTER

INDUCTION OF EXPRESSION OF THIS

GENE BY ADDITIONAL TETRACYCLINE.

SO WE SEE ALREADY AT THE

EARLIEST TIME POINTS, WE

DECREASE PROTEIN EXPRESSION, BY

MINIMAL ACTIVITY, AND mRNA

LEVELS ARE DECREASED WITH THE

CONSIDERABLE DELAY.

AND THIS IS SEEN WITH ALL THREE

REPORTERS WHICH WE WERE TESTED.

AND THEN WE DID THE EXPERIMENT

WHERE WE INDUCED EXPRESSION BY

ADDITIONAL TETRACYCLINE AND THEN

AFTER ONE HOUR, WE ADD TO BLOCK

TRANSCRIPTION AND LOOK AT mRNA

LEVELS, POLYA STAT US AND

PROTEIN LEVELS.

SO AGAIN, WE SEE THAT EARLIEST

TIME ALREADY, WE SEE PROTEIN

SYNTHESIS REPRESSED WITHOUT ANY

EFFECT ON mRNA LEVEL AND ONLY

AS LATER TIME POINTS WE SEE SOME

EFFECT ON POLYA LENT.

THIS IS THIS.

AND mRNA LEVEL, AND EVENTUALLY

IT'S THINKA -- AFTER LONGER

TIME, THE REPRESSION CAN BE

EXPLAINED BY mRNA DEGRADATION

IN THIS CASE.

SO WE CAN REFER THIS RESULT AS

INDICATING THAT AFTER THE

TRANSLATIONAL REPRESSION COMES

FIRST, AND AFTER SOME DELAY,

20-40 MINUTES, THE DEADDALATION

IS TAKING PLACE.

AND SIMILAR RESULTS PUBLISHED

FOR ZEBRAFISH THEY INDICATE THAT

TRANSLATIONAL REPRESSION IS

PROBABLY THE DOMINANT FACTOR OF

MICROR-NA FUNCTION IN EARLY TIME

POINTS.

THIS IS IMPORTANT POINT BECAUSE

SEVERAL PAPERS, SOME COMING FROM

MIT ARGUING THAT DEGRADATION IS

EXPLAINED EVERYTHING AND THAT

THEY KIND OF DEFAULT FASHION OF

MICRORNAs IS mRNA

DEADENYLATION AND DEGRADATION

AND THIS IS HARD TO UNDERSTAND

THAT THIS WOULD BE THE CASE

BECAUSE THERE ARE MANY EXAMPLES

IN THE LITERATURE COMING FROM

NOT GENOMIC STUDIES WHERE ONE IS

LOOKING GLOBALLY AT THE WHOLE

GENOME BUT IN INDIVIDUAL

MRNAs WHEN ONE IS SEEING

MRNAs WHICH ARE REPRESSED BY

MICRORNAs AT THE TRANSLATIONAL

LEVEL WITHOUT CONSIDERING

DEGRADATION OF mRNA.

AND HERE IS PROBABLY IMPORTANT

TO MENTION NOW THAT PERHAPS THE

SAME COMPLEX, CCR4-NOT IS

RESPONSIBLE FOR DEATALATION OF

mRNA AND TRANSLATIONAL

REPRESSION.

Y -- EITHER ACTIVITY OF THIS

THIS COMPLEX REPRESS TRANSLATION

WILL BE DOMINATING ONE AND ONE

COULD AGAIN GO BACK AND FORTH

BETWEEN THIS STRUCTURE OF

ACTIVITIES OF THE SAME COMPLEX.

SO THIS IS JUST WHAT I HAVE BEEN

DISCUSSING.

ANOTHER POINT WHICH I WANT TO

MAKE IS THAT LAYERS OF

INTERACTIONS, THE MICRORNA

ATTRACTING GW182.

GW182 ATTRACTING CCR4-NOT AND

WHO KNOWS HOW MANY FACTORS ARE

WORKING IN BETWEEN.

CREATE A LOT OF POSSIBILITIES TO

REGULATE WHOLE PROCESS.

IF ONE THINKS ABOUT MICRORNA

REGULATION AS A PATHWAY

EFFECTING MORE THAN HALF OF THE

GENES IN THE CELL, THIS IS

PROBABLY NOT UNEXPECTED THAT

THIS COMPLEXITY SUBJECT TO POST

TRANSLATIONAL MODIFICATION IS

THERE TO ALLOW KIND OF GREATER

STAGES OF INHIBITION.

SO, I WOULD LIKE NOW TO MOVE TO

THE SECOND PART, TO REGULATION

OF MICRORNA MEDIATED REPRESSION

IN METABOLISM.

SO I ONLY VERY BRIEFLY WANT TO

MENTION THE WORK WHICH WE DID ON

REGULATION OF MICRORNA

REPRESSION, PER SE.

THERE ARE DOZENS OF REGULATORY

PROTEINS NOW IDENTIFIED, WHICH

MODULATE FOR REPRESSION BY

MICRORNAs.

FACT CHORES ASSOCIATE WITH GW182

PROTEINS, AND BINDS NEXT TO

MICRORNAS AND POSTTRANSLATIONAL

MODIFICATIONS.

SO WE DON'T UNDERSTAND THROUGH

THE BIOLOGY THAT ALL OF THESE

PROTEINS ARE POSTTRANSLATIONAL

MODIFIED AND UBIQUITINATED OR

FOX FOREINATED AND PROBABLY THIS

PLAYS AN IMPORTANT ROLE IN THE

REGULATION.

AND FINALLY, THE REVERSIBILITY

OF MICRORNA REPRESSION.

AT ONE POINT, A COUPLE YEARS

AGO, WHEN WE STARTED TO DO THIS

EXPERIMENT, IT WAS ONLY KNOWN

THAT MICRORNA BIND TO mRNA AND

REPRESS AS A FINAL DECISION AND

ESPECIALLY WHEN MICRORNA WAS

DEGRADED, SO WE REASON THAT THE

PROCESS WOULD BE MUCH MORE

IMPORTANT, MUCH MORE DYNAMIC

WHEN UNDER SOME CONDITIONS,

MICRORNA REPRESSION COULD BE

REVERSED AND YOU COULD GO BACK

AND FORTH.

AND INDEED, WE FOUND THAT IN

HEPATOCYTES, THE MICRORNA182,

WHICH DELIVERS LIVER-SPECIFIC

MICRORNA, IT REPRESSES mRNA

AND CODING AMINO ACID

TRANSPORTER BY BINDING TO 3

PRIME UTR AND THE REPRESSED

MRNA -- WITHOUT ANY

DEGRADATION, ENDOGENOUS mRNA

AND THEN WE NOTICED OUT OF

STRESS, THIS CAN BE ER STRESS,

AMINO ACID STARVATION OR

OXIDATIVE STRESS, HUR, RNA

BINDING PROTEIN IS GETTING OUT

AND BINDS TO THE 3 PRIME UTR AND

IT CAUSES RELIEF OF THE

REPRESSION.

SO mRNA IS DEPARTING FROM THE

P BODY AND RECRUITED TO

POLYSOMES FOR TRANSLATION.

AND JUST WANT TO SHOW ONE

EXAMPLE OF THE DATA.

SO HERE IN A CELL DRAWN IN THE

PRESENCE OF AMINO ACIDS, THE

REPRESSIVE RNAs ARE PRESENT IN

P-BODIES WHEN WE STARVE CELLS

FOR AMINO ACIDS, MRN SAMPLE GONE

AND IT IS ASSOCIATED WITH

POLYSOMES.

SO, THIS PROCESS IS HUR DEPEND

ENTER SO WHEN WE KNOCK DOWN HUR,

THIS DOESN'T HAPPEN AND

TREATMENT -- THISIGED INDICATED

REALLY MICRORNA REPRESSION BEING

RELEASED.

AND HUR TO JUST TO INTRODUCE YOU

BRIEFLY TO THIS PROTEIN,

PROBABLY FAMILIAR WITH IT

BECAUSE ANOTHER LAB IS STUDYING

THIS VERY INTENSIVELY FROM NIHT

IS A PROTEIN WHEN BINDS TO

ELEMENTS IN 3 PRIME UTR AND

STABIZES mRNAs.

IT'S MAINLY LOCIDES IN THE

NUCLEUS BUT SHUTTLES BETWEEN THE

IF YOU CAN PLUS CYTOPLASM AND

TRANSLOCATES FROM THE NUKE

THROWS CYTOPLASM IN RESPONSE TO

STRESS.

AND THIS PROTEIN U.N. REGULATED

AND ACCUMULATES IN THE CYTOPLASM

IN MANY CANCERS.

SO WE COULD ILLUSTRATE THAT WE

CAN MEDIATE THIS PROTEIN, THE

ACTIVITY OF THIS PROTEIN IN THE

ABSENCE OF STRESS AND WE CAN

UNDOUBLE THIS EFFECT FROM STRESS

BECAUSE IT ORIGINALLY WAS VERY

HARD TO PREDICT WHAT THE STRESS

CONDITIONS, WHAT KIND OF CHANGES

THEY WOULD BE USING THEMSELVES.

SO BY USING HUR MUTANTS IN

CANCER CELLS AND CYTOPLASMIC

HUR, WE COULD SHOW THAT LOCATION

OF HUR IS SUFFICIENT FOR THE

EFFECT.

AND MORE SIMPLY, WE ARE

INTERESTED IN A KIND OF HOW THIS

PROTEIN ACTUALLY IS MEDIATING

THIS EFFECT AND WE COULD SHOW

THAT THIS PROTEIN, WHEN BINDING

3 PRIME UTR DISPLACES THE

MICROFROM mRNA AND DOES IT

BECAUSE IT IS OLOGY MERARISES

FROM THE MESSAGE AND HOW EXACTLY

DOES IT, DOESN'T PHYSICALLY

DISPLACE MICRORNA, THIS WE DON'T

KNOW.

I HAVE NO TIME TO SHOW THIS DATE

THAT WAS RECENTLY PUBLISHED BY A

STUDENT IN THE LAB.

SO, I WANT TO FINISH THIS PART

WITH JUST TELLING YOU THIS

INTERPLAY BETWEEN MICRORNA AND

RNA BINDING PROTEINS BINDING TO

DIFFERENT REGIONS IN THE 3 PRIME

UTR IS A VERY COMMON SENTIMENT.

AND THERE ARE DOZENS OF EXAMPLES

OF THIS OF THIS TO DESCRIBE OURS

WAS THE FIRST AND THIS CAN WORK

IN DIFFERENT DIRECTIONS, RNA

BINDING PROTEINS CAN HAVE A

POSITIVE EFFECT ON REPRESSION

AND NEGATIVE AND VICE VERSA.

MICRORNPs CAN HAVE A POSITIVE

OR NEGATIVE EFFECT ON RNA

BINDING PRO DISPENSE THIS CAN BE

DUE TO DIFFERENT MECHANISMS VERY

OFTEN MICRORNA AND RNA BINDING

PROTEINS COMPETE FOR THE SAME

SEQUENCE TO BIND TO.

SO SINCE 3 PRIME UTR IS VERY

OFTEN VERY LONG AND THERE ARE

HUNDREDS OF RNA BINDING PRO

DISPENSE MICRORNAs, YOU CAN

IMAGINE HOW COMMON THIS TYPE OF

COREGULATION WILL BE.

I WANT TO THEN MOVE TO THE

REGULATION OF BIOGENESIS, JUST

BY TELLING YOU THAT THERE ARE

MANY FACTORS WHICH MODULATE

BIOGENESIS MICRORNAs.

THIS PROTEINS INCLUDE PROTEINS

LIKE p53, SMA.

AND HNRNP PROTEINS, KSRP, AND

P68, AND P72 HELICASES AND THERE

ARE MANY, MANY EXAMPLES OF THIS

PROTEINS BINDING TO THE USUALLY

HAIR PINS OF MICRORNA PRECURSORS

EITHER IN THE NUCLEUS AND THEN

THEY EFFECT DOCTORRA PROCESSING

IN A POSITIVE OR NEGATIVE WAY.

OR IN A CYTOPLASM WHERE THEY

EFFECT IN A POSITIVE OR NEGATIVE

WAY PROCESSING.

SO THIS IS TAKEN FROM AN ARTICLE

PUBLISHED SOME TIME AGO IN

COLLABORATION WITH ROBERTO AND

MICHAEL ROSENFELD.

I WANT TO NOW JUST MOVE TO THE

FINAL PIECE OF EXPRESSION I WANT

TO DISCUSS THE REGULATION OF

MICRORNA DECAY.

MICRORNA ARE GENERALLY BELIEVED

TO BE VERY STABLE MOLECULES.

VERY LONG LIVED WITH A HALF-LIFE

EXTENDING INTO DAYS.

THE EXAMPLES OF MICRORNAs

DELIVER AND DO NOT CHANGE THEIR

LEVELS OF THE BASE.

SO, WE ACTUALLY STARTED TO

INVESTIGATE THIS PROBLEM.

WE WERE INTERESTED.

I HAVE A VERY GOOD COLLEAGUE, A

SPECIALIST, RETINA PHYSIOLOGIST

AT A INSTITUTE, AND WE DECIDED

AT ONE POINT TO COMBINE FORCES

AND LOOK AT MICRORNAs WHICH

RESPOND TO LIGHT OR DARK

ADAPTATION.

SO WE DID A SET OF EXPERIMENTS

WHERE WE KEEPING MICE IN LIGHT

OR MOVING THEM AFTER 3 HOURS IN

LIGHT INTO DARK, AND COLLECTING

SAMPLES OF MICE KEPT IN LIGHT OR

IN DARK AND DOING DEEP

SEQUENCING RNA PROTECTION IN

REALTIME PCR AND MICRORNA AND WE

CAME UP WITH A COUPLE OF

MICRORNAs WHICH ACTUALLY

DECREASE DURING THIS THREE HOURS

EVER DARK ABOUT 2 1/2 FOLD.

SOMETHING UNHEARD OF THAT

MICRORNA WOULD BE GOING DOWN SO

RAPIDLY THAT THE TIME.

SO THIS WAS DONE A COUPLE OF

YEARS AGO.

AND PUBLISHED RECENTLY BUT NOW

THERE ARE MORE EXAMPLES OF

MICRORNAs TURNING OVER FASTER.

SO THIS IS JUST RNA PROTECTION

NOW SHOWN THE SAME.

SO WE THEN DID THE KINETICS

COLLECTING RETINA AT HALF HOUR.

AND WE FOUND THAT THIS DECREASED

IS NEVER MORE THAN TWO FOLD AND

IT IS RELATIVELY RAPID WITH

HALF-LIFE OF ABOUT 45 MINUTES.

AND BY PUTTING THE ANIMALS BACK

TO LIGHT, IT WAS A VERY RAPID

INCREASE IN MICRORNA LEVEL.

SO BY DOING A LOT OF

BIOCHEMISTRY AND INHIBITOR

STUDIES, WE COULD SHOW IN DARK,

MICRORNAs GENERALLY DECAY

RAPIDLY AND TRANSCRIPTION OF

THIS MICRORNA REGULATE SAID LOW.

NOW YOU MOVE ANIMALS TO LIGHT,

TRANSCRIPTION IS UP, AND THIS

COMPENSATES FOR THE DECAY.

WE WERE SURPRISED TO FIND OUT

THAT EACH MICRORNA LEVELS IS NOT

REGULATED LIKE LED 7.

IT'S ALL VERY UNSTABLE.

IT HAS THE SAME LEVELS IN DARK

AND LIGHT BUT THIS IS DUE TO

HIGH TRANSCRIPTION AND RAPID

DECAY, WHICH COMPENSATE FOR EACH

OTHER.

SO THIS IS JUST AN EXAMPLE OF

THE EXPERIMENT WHERE WE IN

JECT -- IN ONE EYE USING THE

SECOND EYE AS A CONTROL AND WE

SHOW LED 7 MICRORNA WAS NOT

CHANGING AT LEVELS, IS GOING

DOWN WHEN YOU INJECT THIS AND

DECAYS VERY FAST.

SO THIS, AND WE FOUND SIMILAR

RESULTS WITH SEVERAL OTHER

MICRORNAs WHICH DO NOT GO UP

AND DOWN IN DARK BUT THEY DECAY.

SO WHAT HAPPENS IN THE RETINA IS

THAT MICRORNAs TURN OVER BY

DEFAULT VERY FAST.

AND ONLY WHEN NEED TO INCREASE

MICRORNA LEVEL IN LIGHT, YOU

INCREASE TRANSCRIPTION WHEN YOU

PUT THE LIGHT OFF TRANSCRIPTION

IS DOWN AND THEN OF COURSE THE

LEVEL OF MICRORNA GOES DOWN.

IF WE HAVE SOME BIOLOGICAL

UNDERSTANDING, WHAT IS GOING ON,

ONE OF THE TARGETS OF THIS

CLUSTER WHICH CHANGES THE LEVEL

IN DARK, IT GOES DOWN, CONTROLS

EXPRESSION OF THE GLUTEINATE

TRANSPORTER.

AND IN DARK, THERE IS A

CONTINUED TO RELEASE OF

GLUTAMATE INTO THE FIRST VISUAL

SYNAPSE.

AND YOU NEED TO ACTIVATE THIS

GLUTEINATE VERY FAST BECAUSE

OTHERWISE TO HIGH LEVELS WOULD

BE TOXIC AND YOU INDUCE IN DARK

EXPRESSION OF THIS PARTICULAR

GLUTAMATE TRANSPORTER.

NOW, THE QUESTION IS, SO, THIS

IS -- WE TESTED ALSO PRIMARY

RETINAL GLIOCELLS FROM RETINA.

THERE IS NO TURNOVER.

RETINAL PIGMENTED RECEIVING NO

TURNOVER AND ES CELLS AND NO

TURNOVER WITHIN 10 HOURS OR SO.

SO THE QUESTION, AND THIS IS MY

LAST QUESTION I WANT TO REPEAT

AND DISCUSS.

DO MICRORNAs TURNOVER RAPIDLY

IN ALSO NON-RETINAL NEURONS?

YES, THEY DO.

AND WE LOOKED AT LIFE CULTURES

AND PEOPLE IN CORTICAL NEURONS

AND ALSO NEURONS DERIVED FROM

EAR CELLS AND LOOKED AT

DIFFERENT MICRORNAs.

NEURON-SPECIFIC AND OTHERS.

AND YOU CAN SEE THAT WHEN WE

LOOK AT RELATIVITY COMPOST

SLICES THEN THE NEURONAL

MICRORNAs GO DOWN AND THEY

BLOCK TRANSCRIPTION WITH ALPHA.

WHEN WE LOOK AT HIPPOCAMPAL

STRUCTURES OR NEURONS OF

CORTICAL NEURONS, WE SEE THE

SAME.

AGAIN THEY GO DOWN ABOUT TWO

FOLD.

ONLY TWO FOLD.

WHAT WAS THE MOST INTERESTING

PERHAPS IS THAT THIS TURNOVER IS

ENTIRELY DEPENDENT ON NEURONAL

ACTIVITY.

WHEN WE BLOCK ACTION POTENTIALS,

WITH THE TOXIN, WE BLOCK THIS

TURNOVER AND WE DID CONTROLS

WHERE WE WERE LOOKING AT THE

LEVELS OF mRNAs OR PRIMARY

TRANSCRIPT OF MIKE ROW RNAs.

THOSE ARE NOT AFFECTED BY THIS.

SO YOU NEED FIRING NEURONS AND

ACTIVE NEURONS WITH TURNOVER TO

TAKE PLACE.

AND WE EXTENDED THIS TO THE CELL

DERIVED NEURONS AND HIPPOCAMPAL

NEURONS LOOKING AT THE EFFECT OF

GLUTAMATE OR BLOCKING GLUTAMATE

RECEPTORS.

WHEN WE ADD ADDITIONAL GLUTAMATE

TO NEURONAL CULTURE, WE

ACCELERATE TURNOVER FROM THIS

RATE TO THIS RATE AND THE SAME

IS TRUE FOR HIPPOCAMPAL NEURONS.

WITH WE BLOCK KINE 8 OR

RECEPTORS WITH DIFFERENT

CHEMICAL COMPOUNDS, WE BLOCK

TURNOVER.

WE ALSO HAVE ONE MICRORNA WHICH

BEHAVES IN A COMPLETELY OPPOSITE

WAY.

IT'S TURNOVER DEPENDS ON

BLOCKING ACTIVITY AND THIS

MICRORNA ACTUALLY HAS OPPOSITE

FUNCTIONS AT A MICRORNA IN

GENETICS AND DENDRITIC SPINE

DEVELOPMENT.

SO, HERE IS JUST MY LAST

DISCUSSION SITE.

WHY MICRORNAs WITH TURNOVER SO

FAST NEURONS AND WHY WOULD IT

DEPEND ON NEURONAL ACTIVITY?

THIS DECREASE IN 50% LEVEL MAKES

US THINK THAT WE HAVE SOME DATA

SUPPORTING THIS, THAT ONLY ONE

POOL OF MICRORNA U.S. IS TURNING

OVER.

AND PERHAPS THE ONE WHICH IS

PRESENT AT SYNAPSEIS AND

DENDRITES IS ACTUALLY TURNING

OVER FAST.

THERE IS A RECENT PAPER IN

NATURE AND SCIENCE INDICATING

THAT BDNF IS HAVING AN EFFECT AT

THE SYNAPSE ON AN MICRORNA

TURNOVER.

SO POSSIBLY, IT IS WELL-KNOWN,

WELL ESTABLISHED, THAT

TRANSLATION IS REGULATED IN THE

SYNAPSE.

AND THIS ACTIVATION OF

TRANSLATION SYNAPSE IS ACTIVITY

IS NEEDED FOR CONNECTIVITY FOR

LONG-TERM MEMORY FORMATION.

SO, PERHAPS THE STIMULATION AND

ACTIVITY INCREASES OF

TRANSLATION IN THE SYNAPSE IS

ASSOCIATED WITH REMOVAL OF

MICRORNA, WHICH IS ACTIVE AS AN

INHIBITOR OF TRANSLATION OF THE

SINNANS AND IS THERE A LOT OF

SUPPORT FOR THIS IDEA ALREADY

AVAILABLE.

SO, AN ALTERNATE POSSIBILITY

WOULD BE THAT THE STIMULATION OF

NEURONS ACTUALLY EVENTUALLY

ACTIVATING ALL THE TRANSCRIPTION

AND PERHAPS YOU NEED TO TURN

OVER MICRORNAs THEMSELVES TO

MAKE NEW MICRORNAs AVAILABLE

FOR LOADING INTO NEWLY

TRANSCRIBED MESSAGES WHICH

TRAVEL FROM THE NUCLEUS TO THE

SYNAPSE.

SO THIS IS ALL KIND OF WAITING

STIMULATIONS.

WE HAVE NO IDEA BUT WE ARE

WORKING ON THIS.

WE DON'T KNOW WHAT IS IN THE

TURNOVER.

WE WERE LOOKING AT THE LEVELS OF

COMPONENTS OF MICRORNA MACHINERY

LIKE DICER PROTEIN OR OTHER

PROTEINS.

THOSE DO NOT CHANGE.

PROBABLY SOME KIND OF INDUCTION

OF NUCLEASES WHICH ARE

DEGRADING mRNA.

I THINK I ONLY WANT TO NOW

ACKNOWLEDGE MY COLLABORATORS SO

THOSE SHOWN IN RED HERE ARE

CONTRIBUTED IN A DIFFERENT WAY.

TO THE START I ONLY WANT TO LIST

IN PARTICULAR JACEK KROL

AND -- [ READING ]

WE COLLABORATED WITH MANY

PEOPLE, PARTICULARLY WITH

NAHUM SONENBERG AND OTHER PEOPLE

AND I DON'T KNOW WHETHER I

MENTIONED THIS BUT WE ARE NOT

THE ONLY ONES WORKING IN THIS

FIELD.

THIS IS A VERY COMPETITIVE FIELD

AND THERE ARE MANY STUDIES DONE

BY OTHER LABS VERY OFTEN COMING

TO SIMILAR CONCLUSIONS, WHICH IS

VERY GOOD BECAUSE THERE ARE A

LOT OF CONCRETE MICRORNA

STUDIES.

THE FACT THAT THREE GROUPS

IDENTIFIED CCR4-NOT COMPLEXES AS A

DOWNSTREAM PROMOTOR IS SOMETHING

UNHEARD OF.

WHAT WHICH MAKES US HAPPY.

THANK YOU VERY MUCH.

[ APPLAUSE ]

>> I THINK WE HAVE A FEW MINUTES

FOR QUESTIONS.

IF YOU LIKE TO HAVE A QUESTION,

COME TO THE MICROPHONE.

SYSTEM.

>> SO, VERY NICE TALK.

YOU SHOWED YOU PROVIDED EVIDENCE

THAT IT IS TRANSLATIONAL

DEPRESSION FOLLOWED BY mRNA

DEGRADATION AND I BELIEVE THE

mRNAs YOU'RE LOOKING AT AND

THE MICRORNAs ARE TARGETING

THE 3 PRIME UTR.

BUT IT'S ALSO KNOWN THAT SOME

MICRORNAs CAN REGULATE GENE

EXPRESSION BY BINDING TO THE

CODING VISION.

SO DO YOU THINK A SIMILAR

MECHANISM WOULD BE APPLICABLE TO

THOSE TARGETS THAT ARE REGULATED

BY BINDING OF MICRORNAs TO THE

CODING REEG 71.

>> WE DID NOT ADDRESS THIS

OURSELVES.

BUT A PROFESSOR ANALYZED GENOMIC

AND KEY DATA OF OTHERS AND SHE

CONCLUDED THAT LOCATED IN A

CODING REGION, THERE IS LESS

EFFECT AND DEGRADATION BUT MORE

EFFECT ON TRANSLATION.

ALTHOUGH GENERALLY ITS SITES

HAVE LESS EFFECT IN THE SITES

THAN THE 3 PRIME UTR REGION.

>> I HAD THREE QUESTIONS ABOUT

THE FIRST PART.

SO WHEN YOU'RE USING THE 3 PRIME

RNAs AND YOU SEE THE

TRANSLATIONAL REPRESSION, THERE

ARE ANY CLUES ABOUT WHICH

SUBUNITS OF THE CRR-NOT COMPLEX

KNOCKDOWN EXPERIMENTS

REQUIREMENTS, BESIDES NOT 1?

>> YES, SO NOT 1 IS THE CENTRAL

BECAUSE IT'S ESSENTIAL FOR

COMPLEX.

SO THIS IS SOME DATA FROM

WIGGINS LAB WHO WAS TETHERING

AND HE FINDS THAT IF CAP 1 IS

TETHERED TO POLYA MINUS mRNA

AND INJECTED LOCI, IT INHIBITS

TRANSLATION IN DEPENDENT WAY.

THERE IS A LOT OF KIND OF

CIRCUMSTANTIAL EVIDENCE THAT CAP

1 MIGHT ACT AS INHIBITOR OF

TRANSLATION OR TRANSLATION

INITIATION IN YEAST CELLS COMING

FROM DIFFERENT GROUPS, WHICH

MAKES US THINKING ABOUT CAP 1 AS

A POSSIBLE FACTOR IN THIS.

IF ONE LOOKS AT CAP 1 -- WE SEE

NOTHING APART FROM CATALYTIC

DOMAIN OF THE ENZYMES.

SO WE ARE LOOKING INTO THAT.

WE DON'T KNOW.

WE DON'T KNOW.

>> ANOTHER QUICK THING I WAS

WONDERING ABOUT IS THE GW

REPEATS THAT CONTAINS SEREINS

AND 319S.

IT SEEMS LIKE THE 3 PAL C2D.

ANY EVIDENCE OF PHOSPHORYLATION

THAT MIGHT EFFECT --

>> WE DID NOT LOOK OURSELVES.

BUT IN A MOUSE PROTEOME, THE

SAMPLES IDENTIFIED THE GW182

PROTEINS.

NONE OF THEM FALLS INTO THE

SEREIN OR 3A 19S NEXT TO

TRYPTOPHANS WE WERE LOOKING AT.

THIS WAS A VERY ATTRACTIVE IDEA

BECAUSE THERE IS A POSSIBILITY

OF THE REPEAT TO C NOT 1 BUT WE

HAVE NO IDEAS OF THAT.

>> I HAVE A QUESTION REGARDING

TERMINAL MICRORNAs.

A COUPLE OF RECENT STUDIES THAT

THERE IS A MACHINE THAT

UBIQUITIN RATES RNA AND THAT IS

IMPORTANT TO TURNOVER.

CURIOUS IF SOMETHING HAPPENS IN

MAMMALIAN CELLS?

>> YES.

SO THERE IS A LINK.

THERE ARE SOME STUDIES COMING

FROM PHIL'S LAB INDICATING THAT

WHEN MICRORNAs BASE PAIRS TO

THE TARGET, HIGH

COMPLIMENTIARITY, SO RESEMBLES

siRNA RATHER THAN MICRORNA, IT

IS RELATED TO THE 3 TIME END AND

THEN DEGRADED.

SO DELA NATION MIGHT BE A STEP

IN DEGRADATION RNA.

WE DON'T KNOW HOW THIS IS IN

NEURONS.

WE WERE DOING DEEP SEQUENCING IN

MICRORNAs UNDER CONDITIONS

WHERE YOU HAVE TURNOVER, YOU

DON'T HAVE TURNOVER.

BUT WE DIDN'T GET A CLEAR ANSWER

FROM THAT NEW KNOW WHAT THE

NUCLEUS -- EXESOME OR SOME

OTHER --

>> OKAY.

SO, SO FAR, THERE ARE TWO

CLASSES OF ENZYMES IMPLICATED IN

THE DEGRADATION.

IN PLANTS, THOSE ARE 3 PRIME 5

PRIME EXONUCLEASES, SNPS 2 AND

3.

I THINK YOU HAVE TO KNOCK THEM

DOWN, 3 OF THEM, TO SEE THE

PHENOTYPE.

IN MAMMALS -- NO WORK IN

MAMMALS.

BUT THERE IS WORK DONE IN C

ELEGANS WHICH IMPLICATES 1 AND

2.

IN A 3 PRIME TO 5 PEOPLE

DIRECTION DEGRADATION.

SO THEY ARE COMPLETELY OPPOSITE

ENZYMES OPPOSITE ACTIVITY

IMPLICATED.

WE DON'T KNOW WHAT IS IT

ACTUALLY IN MAMMALS.

>> IS THERE A RECEPTION IN THE

LIBRARY AND EVERYBODY IS WELCOME

AND THANK YOU VERY MUCH.

[ APPLAUSE ]