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>> 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 ]