>> GOOD AFTERNOON.
BEFORE I FORGET, THE RECEPTION 
AFTER THIS TALK IS GOING TO BE 
IN THE LIBRARY, SO YOU CAN GO 
THROUGH THESE DOORS AND IT'S 
RIGHT THERE.
SO YOU BETTER STAY UNTIL THE END
BECAUSE THERE'S COOKIES, I 
THINK.
GOOD AFTERNOON AND WELCOME TO 
TODAY'S WEDNESDAY AFTERNOON 
LECTURE SERIES ON THE TUESDAY, 
ON BEHALF OF THE WASHINGTON AREA
EAST INTEREST GROUP IS IT MY 
PLEASURE TO INTRODUCE TODAY'S 
SPEAKER, DR. SUSAN WENTE FROM 
VANDERBILT SCHOOL OF MEDICINE.
HE RECEIVES BACHELORS IN 
CHEMISTRY FROM UNIVERSITY OF 
IOWA.
AFTER A SHORT BUT PRODUCTIVE 
POST DOC, SUSAN JOINED RO KA 
FELLER DWRUFRT WORK ON WHAT 
BECAME THE FOCUS OF HER RESEARCH
EVER SINCE T NEWLY YAR PORE 
COMPLEX.
IN 1993, SHE BECAME A FACULTY 
MEMBER OF THE WASHINGTON 
UNIVERSITY SCHOOL OF MEDICINE IN
ST. LOUIS WHERE SHE CONTINUED TO
PUBLISH PAPERS ON THE STRUCTURE 
OF NEWLY YAR POOR AND EXPORTS.
IN 2002 SHE WAS APPOINTED CHAIR 
AT VANDERBILT UNIVERSITY SCHOOL 
OF MEDICINE AND SINCE 2009 SHE'S
BEEN AN ASSOCIATE VICE 
CHANCELLOR AT VANDERBILT SPP 
SHE'S HEALTH HELD NEW MORE ROUS=) 
POSITIONS, BEEN A MEMBER OF 
VARIOUS STUDY SECTIONS, ADVISORY
BOARDS, EDITORIAL BOARDS JUST TO
NAME A FEW.
AWARDS WON JOHN EXTON AWARD.
THERE IS NO DOUBT THAT SUSAN'S 
RESEARCH HAS AN INCREDIBLE 
IMPACT ON OURç UNDERSTANDING OF 
NEW KRI YAR CYTOPLASMIC 
TRAFFICKING AND PERHAPS WHAT IS 
MOST IMPRESSIVE THAT S THAT SHE 
CONTINUE TOSS PUBLISH HIGHLY 
INFLUENTIAL STUDIES WHILE 
SERVING SCIENTIFIC COMMUNITY AND
RAISE THE WEIGHT TO BE GIVEN 
CHILDREN.
WITH THIS I UH WOULD LIKE TO 
INVITE SUSAN TO THE PODIUM P P 
TITLE IS BEYOND NUCLEAR PORES 
FROM mRNA EXPORT TO TRANSLATION.
>> THANK YOU VERY MUCH AND THANK
YOU TO ALL OF YOU BEINGkOñrç HERE 
TODAY ON MY REKRED CALL WALLED 
VISIT WHERE THERE'S NO SNOW AND 
MAYBE JUST THREAT OF A 
THUNDERSHOWER, BUT QUESTION 
SURVIVE THAT.
IT'S A PRIVILEGE TO BE HERE TO 
TALK ABOUT THE WORK THAT WE'RE 
DOING IN MY LABORATORY.
MY LAB IS FOCUSED ON NUCLEUS 
CYTOPLASMIC TRANSPORT A 
FUNDAMENTAL ASPECT OF ALL CELL 
BIOLOGY.
TO START OFF MY TALK BECAUSE I 
WAS INVITED HERE AS PART OF THE 
YEAST CONSORTIUM GROUP AND 
BECAUSE MY NEW-FOUND EXPANDING 
ADMINISTRATIVE ROLES ONE THEME 
THAT I HOPE YOU TAKE HOME IS 
THAT YOU CAN WORK WITH A MODEL 
SYSTEM ARK BUDDING YEAST, AND 
MAKE FUNDAMENTAL INSIGHTS INTO 
HUMAN HEALTH.
THE STRATEGY THAT MY LABORATORY 
HAS TAKEN ASgobw3 ITS OVERALL 
FRAMEWORK OVER THE LAST 18 YEARS
IS BY STUDYING HOW NORMAL CELLS 
WORK AND DETERMINING WHAT PARTS 
ARE IMPORTANT THAT WE CAN 
EVENTUALLY HAVE INSIGHTS INTO 
WHAT IS BROKEN OR HOW WHAT IS 
BROKEN CAN BE FIXED IN A 
DISEASED CELL.
I WILL ALSO ARGUE THROUGHOUT 
THIS TALK AT DIFFERENT POINTS 
THAT A DOING BOTH OF THESE TYPES
OF STUDIES IN PARALLEL IS 
ESSENTIAL AND THAT THEY ARE 
INTERDEPENDENT.
NOW, IN TERMS OF, UM, NEWICALLY 
YUM CYTOMRAZ MANY TRANSPORT, 
THIS IS TISSUE GROAN IN CULTURE.
THE GENOMIC MATERIAL IS INCASED 
IN A COMPARTMENT OR A ROOM IN 
THE CELL TERMED THE NUCLEUS.
THE NEW KRI YUS IS REALLY, COULD
BE CONSIDERED THE CONTROL CENTER
OF THE CELL.
AND THAT IF YOU CAN CONTROL THE 
NUCLEUS, YOU COULD CONTROL ALL 
CELLAR FUNCTION.
THIS ASPECT OF INCASING THE DNA 
WITHIN THIS NUCLEAR COMPARTMENT 
SEPARATES IT FROM THE SIDE OF 
PROTEIN SYNTHESIS IN1Eç THE 
CYTOPLASM.
TO BE ABLE TO CONTROL DIFFERENT 
ASPECTS OF CELLAR PHYSIOLOGY, 
MACROMOLECULES HAVE TO BE 
EXCHANGED BETWEEN THESE TWO 
COMPARTMENTS.
MOVING IN AND OUT OF THE NUCLEUS
HAS BEEN A FUNDAMENTAL AREA OF 
RESEARCH AND YOU COULD CONSIDER 
THE ACTUAL MECHANISM REQUIRES 
SPECIAL DOORS IN THE NUCLEAR 
ENVELOPE WHICH WE REFER TO AS 
NUCLEAR PORE COMPLEXES.
THIS INCLUDE, ONE, THAT SMALL 
MACROMOLECULES THAT DIFFUSE 
THROUGH THESE DOORS BACK AND 
FORTH, THIS MAKING THIS 
DISTRINKET FROM THE TRANCE 
LOCATION, FOR EXAMPLE, DISTINCT 
FROM THE ER MEMBRANE ACROSS THE 
OTHER MEMBRANES AND MIE TOE CON 
KRI Y'ALL MEMBRANE.
SIMILAR TO NOSE OTHER TRANCE 
LOCATION MECHANISMS LARGER 
MACROMOLECULES REQUIRE A 
FACILITATED SIGNAL DEPENDENT 
MECHANISM.
NOW, IN TERMS OF THE AMOUNT OF 
TRANCE LOCATION AND THEkO TYPES OF
FACTORS TRANCE LOCATED BETWEEN 
THE NUCLEUS AND THE CHI TOE 
PLASM, AS I REFER TO THE -- ANDd8 
BASED UPON THE REACTION OF 
[INDISCERNIBLE] THE RESULTING 
MESSENGER RNA AND N THE FORM OF 
[INDISCERNIBLE] SUB UNITS AND 
SHUTTLING PROTEINS ARE EXPORTED 
OUT OF THE NUCLEUS TO THE 
CYTOPLASM.
ALL OF THE PROTEINS TRANSLATED 
INTO THE CYTOPLASM HAS HAVE TO 
BE IMUH PORT INTO THE NUCLEUS IN
ORDER TO EXECUTE THEIR FUNCTION.
THERE'S ESTIMATES BASED UPON THE
AMOUNT OF TRANSPORT THAT NEEDS 
TO HAPPEN TO MAINTAIN NORMAL 
CELLAR PHYSIOLOGY THAT THERE'S 
ONE TO TWO MILLION MOLECULES 
EXCHANGED PER SECOND WITHIN A 
NORMAL YOU CAR YO TICK CELL.
SO, IN TERMS OF THE 
SIGNAL-DEPENDENT MECHANISMS USED
TO TRANCE LOCATE, THIS RELIES 
UPON TRANSPORT RECEPTORS AND 
ENERGY TO MOVE THROUGH THOSE 
NUCLEAR PORE COMPLEXES.
IF THIS IS A PROTEIN WHICH IS 
DESTINEDç FOR THE NUCLEUS, ON ITS
SURFACE THERE'S AN AMINO ACID 
SEQUENCE WHICH COULD BE 
CONSIDERED A KEY WHICH GETS 
RECOGNIZED BY THAT TRANSPORT 
RECEPTOR FOR TRANSPORT INTO THE 
NUCLEUS, AND IN THE PARADIGM 
THAT FOLLOWS THE TRANCE LOCATION
MECHANISM ACROSS THE ENDOPLASMIC
RETICUL;-gñr IN TERMS OF HOW THESE 
KEYS ARE RECOGNIZED I'LL REFER 
TO THEM AS BEING REFERRED TO BY 
A MAILMAN THAT CAN DIRECT THEM 
THEIR PROPER CELLAR DESTINATION.
IN THIS CASE THAT WOULD BE 
IMPORT INTO THE NUCLEUS.
NOW, THE DOORS THAT ARE THE SOLE
SIGHT FOR NEWICALLY OWE CYTOMRAZ
I CAN TRANSPORT HAVE BEEN 
STUDIED FOR MANY YEARS.
SO THIS IS AN EXAMPLE OF A THIN 
SECTION ELECTRON MICROGRAPH 
WHERE YOU CAN SEE THE OUTER 
NUCLEAR ENVELOPED FUSED TO THE 
INNER AND IN THIS PASS ONLY WAY,
THIS PROTEIN DENSE MATERIAL 
WHICH ARE ACTUALLY THOSE NUCLEAR
PORE COMPLEXES THAT ARE 
PROVIDING THE CONDUIT FOR 
MESSENGER RNAs TO MOVE OUT AND 
PROTEINS TO MOVE IN.
THESE ASSEMBLIES ARE VERY LARGE 
COMPLEXES WHICH ARE NOW KNOWN TO
BE MADE UP FOR 30 DISDISTINCT 
POLYPEPTIDES.
WHY HAVE SEPARATED TRANSLATION 
AND THE PRODUCTION OF PROTEINS 
IN THE CYTOPLASM FROM THE 
PROTECTION OF THE MESSENGER RNAs
IN THE DRY CRY TOE PLASM?
THIS ALLOWS THE YOU CAR YO TICK 
CELL ADDITIONAL REGULATIONS IN 
TERMS OF TRANSMITTING SIGNALS 
BETWEEN THESE COMPARTMENTS.
IT ALSO ALLOWS REGULATION AT 
MULTIPLE DIFFERENT LEVELS IN 
TERMS OF THE COMPONENTS.
THE CARGO THAT'S BEING 
TRANSPORTED THROUGH THE NUCLEAR 
PORE COMPLEX SO IT ONLY EXITS OR
ENTERS AT SPECIFIC TIMES.
IT'S ALSO TRUE THAT THEKo 
RECEPTORS CAN ALSO BE CONTROLLED
EITHER BY AT DIFFERENTIAL 
EXPRESSION OR BY POTENTIALLY 
SEQUESTERING THEM IN DIFFERENT$x 
WAYS.
FINALLY, THERE ARE INCREASING 
EXAMPLES OF HOW THE NUCLEUS PORE
COMPLEX ITSELF COULD BE 
CONTROLLED TO ALLOW REGULATED 
TRANSPORT AT DIFFERENT PHASES 
EITHER DURING STEM CELL 
DIFFERENTIATION OR DURING AGING.
LINKING THIS FURTHER TO THE FACT
THAT TRANSPORT REGULATION IS A 
NORMAL CELLAR PHYSIOLOGICAL 
PROCESS THAT COULD BE 
POTENTIALLY IMPACTED IN 
DIFFERENT DISEASE MECHANISMS, IT
IS WELL DOCUMENTED BY A NUMBER 
OF DIFFERENT LABORATORIES THAT 
POTENTIALLY DURING CANCER CELL 
GROWTH THE NUCLEUS CYTOPLASMIC 
DYNAMICS COULD BE ALTERED.
THIS SHOWS AN EXAMPLE OF THE 
LOCALIZATION OF P 53 IN THE 
NUCLEUS IN THE NORMAL CELL.
HOWEVER IN A CANCER CELL THE 
LOCALIZATION IS ALTERED.
INCREASING REPORTS WITH THE 
SEQUENCING OF THE HUMAN GENOME 
OF DEVELOPMENTAL DISORDERS THAT 
HAVE BEEN LINKED GENETICALLY TO 
GENES AND CODING FACTORS THAT 
ARE PART OF THE NUCLEAR CORE 
COMPLEXES OR PART OF THE 
TRANSPORT MACHINERY.
IN PARTICULAR THE TRIPLE A 
SYNDROME HAS BEEN LINKED TO 
MUTAUdJju IN CORE COMPLEX 
PROTEIN.
ANOTHER EXAMPLE IS OF FLYER 
SYNDROME WHERE IN A 
TRANSCRIPTION FACTOR HAS 
SPECIFICfá TRANSCRIPTION FACTORS 
IMPORT INTO THE NUCLEUS.
THE OTHER VERY CLASSIC EXAMPLE 
TO KIND OF SET THE FRAMEWORK FOR
YOU IN TERMS OF THE IMPORTANCE 
OF UNDERSTANDING NEWICALLY OWE 
CYTOPLASMIC TRANSPORT IS HOW 
VIESHSS CAN PILOT THESE PATHWAYS
IN ORDER TO ENABLE THEIR 
REPLICATION AND MRIF RATION 
WITHIN THE CELL.
IN TERMS OF MY LABORATORY'S 
APPROACH TO STUDYING THIS FUND 
MENTAL CELL BIOLOGICAL PATHWAY, 
WE'VE REALLY TAKEN AND FOCUSED 
ON AT LEAST THREE DIFFERENT 
QUESTIONS OVER THE YEARS.
ONE OF THOSE QUESTIONS INVOLVE 
WHAT IS THE MECHANISM OF NUCLEAR
PORE COMPLEX ASSEMBLY?
THIS SHOWS YOU TO FOOUZ FUSION 
OF INNER AND OUTER MEMBRANE AND 
THE ASSEMBLY OF A COOR TO AN 
VERSION OF HOW THESE 30 
DIFFERENT PROTEINSñr ASSEMBLE INTO
THAT PORE.
I LAST GAVE A TALK AT NIH WHEN I
WAS ON STUDY SECTION DURING ONE 
TRIP AND ACTUALLY SPENT THAT 
SEMINAR TALKING ABOUT THE 
MECHANISM OF NUCLEAR PORE 
COMPLEX ASSEMBLY.
HOW DOES CAR GROW TRANCE LOCATE?
THE TRANSPORT RECEPTORS BOUND 
FOR CARGO DOC AND MEDIATE TRANCE
LOCATION TO THE CYTOPLASMIC FACE
TO RELEASE CARGO.
j THAT TRANCE LOCATION 
WHAT.
MECHANISM THROUGH THIS NUCLEAR 
PORE COMPLEX?
TODAY I'LL TALK ABOUT A FEW KEY 
STEPS DURING THAT TRANCE 
LOCATION MECHANISM FOR THE 
EXPORT OF MESSENGER RNA.
THE THIRD QUESTION IS WHAT ARE 
THE POTENTIAL CONNECTIONS 
BETWEEN NUCLEAR TRANSPORT AND 
THE REGULATION OF GENE 
EXPRESSION AND THEN OVERALL HOW 
DOES THESE IMPACT DISEASE AND 
DEVELOPMENT?
THE APPROACHES THAT WE'VE USED 
TO ANSWER THESE QUESTION HAVE 
BEEN MULTIFACETED AND AS I'VE 
ALREADY REFERRED TO, ONE OF OUR 
FAVORITE MODEL SYSTEMS FOR, IF 
YOU WANT TO SAY DISCOVERING THE 
DOORS THE KEYS AND THE DELIVERY 
4pRQ(RP'ISMS, HAS BEEN USING THE 
BUDDING YEAST.
THIS SHOW USE GFP TAGGED NUCLEAR
PORE COMPLEX PROTEIN EXPRESSED 
IN BUDDING YEAST WHERE YOU SEE 
THIS PERIPHERAL LOCALIZATION.
WE'VE ALSO EXTENDED STUDIES TO 
TISSUE CULTURES HUMAN CELL 
SYSTEM, AND MORE RECENTLY 
EXTENDED THEM TO USING THE 
ZEBRAFISH MODEL SYSTEM.
I'LL SHOW YOU ILLUSTRATIONS OF 
THE CONSERVATION OF THE 
MECHANISMS AND THE PROCESSES 
THAT WE'VE DISCOVERED IN ALL OF 
O THESE SYSTEMS DURING MY TALK 
TODAY.THESE SYSTEMS DURING MY TALK 
TODAY.
AS I REFERRED TO I'LL FOCUS ON 
TRANSPORTATION OF THE mRNA LIFE 
CYCLE.
THAT WAS ROE FLEKTED IN THE 
TITLE OF MY TALK IN TERM OF mRNA
EXPORLT TO TRANSLATION.
IN TERMS OF THIS ENTIRE LIFE 
CYCLE OF A MESSENGER RNA THERE 
HAVE BEEN MANY REPORTS FROM 
DIFFERENT LABORATORIES ABOUT HOW
DIFFERENT STEPS ALONG THIS 
PATHWAY ARE FUNCTIONALLY COUPLED
TO ONE ANOTHER SUCH THAT DURING 
TRANSCRIPTION THERE'S PROCESSING
AND ASSEMBLY OF A PROTEIN RNA 
STRUCTURE SUCH THAT AT THE END 
OF TRANSCRIPTION AND PROCESSING 
THERE IS A MESSENGER RNP THAT'S 
PROPERLY POLLY IDENTITYLATED AND
CAPPED.
IT HAS A FREEN COHORT ON IT 
RECOGNIZED BY TRANSPORT RECEPTOR
FOR TARGETED TO THE CORE COMPLEX
AND TRANCE LOCATION.
WHAT'S ALSO BEEN DOCUMENTED INñr 
SEVERAL ELEGANT STUDIES IS THAT 
SOME OF THE PROTEINS THAT ARE 
RECRUITED DURING THESE NUCLEAR 
EVENTS ACTUALLY DIRECT THE FATE 
OF THAT xDMRNP ONCE IT REACHES THE
CYTOPLASM.
SO IN OTHER WORDS WHETHER OR NOT
IT'S DIRECTED FOR TRAFFICKING TO
SUB CELLAR DESTINATIONS.
WHETHER IT'S DIRECTED FOR 
TURNOVER AND DEGRADATION OR 
WHETHER FOR MEDIATE TRANSLATION.
THE MESSENGER RNA BINDING 
PR>  PLAY CRITICAL ROLES IN 
MEDIATING AND REGULATING STEPS 
ALONG THIS ENTIRE PATHWAY.
IN TERM OF THE QUESTIONS I'M 
GOING TO ADDRESS IN MY TALK 
TODAY, I'M GOING SPEND THE FIRST
HALF OF THE LECTURE TALKING TO 
YOU ABOUT RECENT INSIGHTS AND 
KIND OF OUR OVERALL MECHANISM 
FOR HOW çMACROMOLECULES AND 
MESSENGER RNAs ARE DIRECTIONALLY
TRANSPORTED THROUGH THE NUKE RAR
PORE COMPLEX.
WHAT ARE THE FACTORS THAT 
REGULATE THE EXIT OFç MRNPs 
THROUGH THIS NUCLEAR PORE 
COMPLEX?
SECONDLY, I'LL TALK TO YOU ABOUT
HOW POTENTIALLY THESE EVENTS 
THAT ARE HAPPENING AT THE 
NUCLEAR PORE COMPLEX IMPACT 
CYTOPLASMIC FATE OF THOSE 
MESSENGER RNPs.
NOW, EVERYCIENTIFIC STORY 
UNDERfá PINNING TO MANY YEARS OF 
WORK OF THE PEOPLE IN THE 
LABORATORY AND THE WORK I'LL 
TALK ABOUT TODAY REALLY SPANS 
THE ENTIRE LENGTH OF TIME THAT 
I'VE BEEN DOING RESEARCH SINCE I
LEFT THE GLOBAL LABORATORY.
IN PARTICULAR T STORY THAT I 
WILL START OUT WITH AND BEGIN 
WITH WAS WITH ONE OF MY FIRST 
GRADUATE STUDENTS, ROB.
HE IS NOT DE DEAD, THIS IS THE 
TIME HE SPENT IN MY LABORATORY.
HE'Sç GOT PLATES STACKED ALL THE 
WAY UP AND THE FIRE MARSHALS -- 
HE TOOK SOME KEY STEPS FROM 
STARTING FROM A SET OF NUCLEAR 
PORE COMPLEX GENES AND LAUNCHED 
INTO STUDYING ESSENTIAL PORE 
COMPLEX FACTORS.
THE NEXT SET OF PEOPLE WHO MADEok 
ADVANCES -- AND THEN I'LL GO ON 
TO TELL YOU HOW THESE STUDIES 
ALL SO KO NEKT TO THE CURRENT 
WORK OF THREE PEOPLE IN MY 
LABORATORY.
SO IN TERMS OF ROB MURPHY'S 
INITIAL WORK IN MY LABORATORY, 
ROB USED YEAST GENETICS STARTING
WITH A MUTANT IN A NUCLEAR PORE 
COMPLEX GENE TO IDENTIFY AN 
ESSENTIAL mRNA EXPORT FACTOR.
WE GAVE THIS A VERY POOR NAME, 
GLEE ONE, IT MEANS NOTHING OTHER
THAN AU GENETIC -- DURING TRE 
SEPGS YOU CAN ASK ME WHAT THAT 
ACTUALLY MEANS.
HE IDENTIFIED THIS ESSENTIAL 
GENE IN YEAST AND IT LOCALIZES 
AROUND THE NUCLEAR RIM, IT'S A 
NUCLEAR PORE COMPLEX AND USING 
THAT WE WERE ABLE TO IDENTIFY A 
HUMAN OR SEW LOG OF THAT YEAST 
GENEu! WHERE IN THE C TERMINAL 
REGIONS ARE VARY FAVORLY HIGHLY 
CONSERVED GIVEN THE EVOLUTIONARY
TIME SPAN AND ALSO LUKEIZED 
ALONG THE NUCLEAR RIM ALTHOUGH 
THERE ARE TWO SPLICED VERSIONS 
ONE MORE PREDOMINANT IN THE 
CYTOMRAZ MRAZ M THE OTHERç AROUND
THE NUCLEAR RIM.
BOTH OF WHICH ALSO HAVE A 
SPECIFIC DOCKING CITE ON THE 
CYTOPLASMIC FACE OF THE NUCLEAR 
PORE COMPLEX.
THESE ARE ESSENTIAL mRNA EXPORT 
FACTORS.
WE CAN DOCUMENT THAT WITH YEAST 
MUTANTS AND ALSO HUMANITY UH 
SHOE CULTURE CELLS USING MIRNA.
THIS IS THE WESTERN BLOCK 
DOCUMENTING THAT WE HAVE KNOCK 
DOWN OF HUMAN GLEE ONE AND WE 
CAN GET ADD BACK BY A RESISTANT 
EXPRESSING RESISTANT CLONES OF 
THE ALTERNATIVELY-SLICED 
VERSIONS.
WE CAN ASSAY BY HIGH 
BERNIZATION.
THIS SHOWS THE TWO TRANSFECTEDED
CELLS IN THIS PANEL VERSUS A 
NON-CELL HERE.
THIS ST LOCALIZATION OF THE 
POLLY IDENTITYLATED RNA.
IN THE NON-CELL WHERE GLEE ONE 
LEVELS ARE NORMAL, YOU CAN SEE 
THAT THE POLLY [INDISCERNIBLE] 
RNA BOTH IN THE CYTOPLASMç AND 
NUCLEUS REPRESENTING NORMAL 
EXPORT VERSUS IN THESE CELLS AND
WHERE WE HAVE KNOCK DOWN OF THE 
ENDOGENOUS GLSHGS GLOOE ONE A 
ISOFORM, YOU SEE ACCUMULATION OF
POLLY IDENTITYLATED RN NASHGS 
THE NUCLEUS AND THEokç CYTOPLASMIC 
LEVELS DROP.
THIS IMPLICATED GLEE ONE AS 
BEING AN ESSENTIAL EXPORT FAK 
FORIN YEAST AND IN HUMAN CELLS, 
BUT ASIDE FROM THE FACT THAT IT 
LOCALIZED AND BOUND TO A 
SPECIFIC NUCLEAR CORE COMPLEX 
PROTEIN ON THE CYTOPLASMIC 
FILAMENTS, WE DID NOT KNOW WHAT 
FUNCTION IT EXECUTED.
TO TRY Eu! TO REVEAL THAT, ROB DID
A SECOND GENERATION GENETIC 
SCREEN, AND THIS WAS MUCHuA$@&EI
COMMITTEE WHO SUGGESTED HE DID 
BIOCHEMISTRY.
THE GENETICS TOLD US WHAT 
BIOCHEMISTRYokkO INDUSTRY WE NEEDED 
TO DO IN THE LONG RUN.
GENETIC SCREEN WITH THIS GLEE 
ONE MUTANT ACTUALLY IDENTIFIED 
ALL OF THE ENZYMES THAT ARE 
REQUIRED FOR THE PRODUCTION OF 
[INDISCERNIBLE] PHOSPHATE IN 
YEAST CELLS.
IP 6 FOR SHORT IS A SOLUBLE 
[INDISCERNIBLE] PHOSPHATE WHERE 
IT'S PHOSPHORYLATED IN  ON ALL 
SIX POSITIONS.ON ALL 
SIX POSITIONS. ON ALL SIX
POSITIONS. ON ALL SIX 
POSITIONS.ON ALL SIX 
POSITIONS.
WE IDENTIFIED IN THIS GENETIC 
SCREEN A DUA FUNCTION
SCREEN A DUAL UH FUNCTI FUNCTIOFUNCTION 
KINASE 
AND THIS KINASE PHOSPHORYLATED 
IP 5 TO IP 6.
THIS GENETIC SCREEN WAS REALLY 
QUITE SURPRISING AND IT WAS TOOK
SEVERAL YEARS TO SORT OUT.
WHEN WE ACTUALLY DID THE FIRST 
CLONING AND IDENTIFIED ONE OF 
THE MUTANT ALLELES ASç PLC 1, 
THIS WAS NOT HA MOLL GUS TO 
ANYTHING IN THE DATABASE.
WE LET IT SIT IN THE FREEZER FAR
COUPLE OF YEARS UNTIL WE WERE 
CONNECTEDçó WITH JOHN YOSHG AT 
DUKE UNIVERSITY WHO LET US KNOW 
HE WAS VERIED IN THIS DOWNSTREAM
PRODUCTS.
WE HAD INCREDIBLY PRODUCTIVE 
COLLABORATION IN TERMS OF MY 
LABORATORY GENERATING MUTANTS 
AND HIS LABORATORY BEING ABLE TO
HELP US DISSECT THAT THE GENES 
WE HAD ACTUALLY IDENTIFIED FOR 
TEN VOOIMS REQUIRED FOR THE 
PRODUCTION OF IP 6.
THIS SHOWS YOU THAT THOSEç THAT 
LACK IP 6 ACCUMULATE POLLY 
IDENTITYLATED RN NASHGS THE 
NUCLEUS.
THEREFORE THIS IS REQUIRED FOR 
MESSENGER RNA EXPORT.
JUST AS A SIDE LIGHT, THIS WAS 
THE FIRST IDENTIFICATION OF 
ENZYMES THAT COULD PRODUCE THESE
SOLUBLE [INDISCERNIBLE] 
THEMSELVES.
MANY LABORATORIES HAVE GONE ON 
TO IDENTIFY ADDITIONAL IN MANY 
OTHER ORGANISMS THAT IDENTIFY 
THESE.w3
WHERE THERE ARE TWO PHOSPHATES 
ON ONE POSITION AND MORE SO HAVE
GONE ON TO IDENTIFY CELLAR 
FUNCTIONS AND SOME OF THESE THIS
Ñ CONDUCT AT NIH IN 
TERMS OF CHROME CHROME TONE 
REMODELLING.
I TOLD YOU WE IDENTIFIED GLEE 
ONE AND THAT WE IDENTIFIED IT 
REQUIRES THE PRODUCTION OF 
[INDISCERNIBLE] PHOSPHATE OR IP 
6.
WHY IS THAT?
SO WORKING FROM REPORTS OF OTHER
FACTORED THAT BEEN IDENTIFIED TO
FIND IP 6 ON ADAR, WE FOCUSA
ON IP 6 POTENTIALLY BINDING 
DIRECTLY TO GLEE ONE AND WE 
COMPARED THE SEQUENCE OF GLEE 
ONE FROM MULTIPLE DIFFERENT 
ORGANISMS AND BASED UPON THE 
COMPARISON OF THE RESIDUES THAT 
ARE REQUIRED, WERE ABLE 
IDENTIFIED CONSERVED RESIDUES.
WHEN THOSE AREñ ALTERED COMPARED 
TO WILD TYPE PROTEIN, WE COULD 
DEFINITIVELY CONCLUDE THAT IP 6 
BINDS DIRECTLY TO GLEE ONE.
THIS IS A EQUAL [INDISCERNIBLE] 
BINDING ASSAY AND WE COULD FIND 
THAT GLEE ONE WILD TYPE 
IDENTIFIES OF APPROXIMATELY 95 
NANO MOLAR AND MAKING MUTANTS 
SEVERELY INHIBITS THE BINDING TO
IP 6.
INTEED, THESE MUTANTS THAT LACK 
IP 6 BINDING FEE KNOW COP O PI 
UH THE MUTANTS THAT LACK IP 6 
PRODUCTION.
AGAIN, SAYING THAT GLEE ONE IS 
THE TARGET FOR IP 6 IN mRNA 
EXPORT.
SO NOW WE HAVE TWO FACTORS WHICH
COME TOGETHER, BIND TO EACH 
OTHER AND EXECUTE A CRITICAL 
STEP IN mRNA EXPORT.
BUTñr WHAT STEP?
THIS LED US TO ANOTHER SET OF 
GENETIC STUDIES, IMPLICATING 
THEM AND REGULATING PARTICULAR 
DEAD BOXED PROTEIN THAT'S 
REQUIRED IN mRNA EXPORT.
SO WHAT ARE DEAD BOX PROTEINS?
AT THIS POINT YOU'RE ALL myw3GOING, 
DOSH GO YOU'RE SAYING A LOT OF 
DIFFERENT FACTORS BUT THE ONLY 
THING YOU TO REMEMBER ARE GLEE 
ONE, IP 6 AND DEAD BOX PROTEINS.
IN TERMS OFç TAED BOX PROTEINS 
THIS, IS A VERY LARGE FAMILY OF 
CONSERVED ENZYME WHICH IS EACH 
OF THOSE COLORED CIRCLES IN HERE
REPRESENT THE FUNCTIONAL LINK 
FOR A DIFFERENT FAMILY MEMBER.
THERE ARE PROBABLY 25 IN YEAST 
AND GREATER THAN 38 IN HUMAN 
CELLS.
SOME OF THEM HAVE HAD EFFECTORS 
IDENTIFIED.
EACH PROTECTED TO HAVE 
RNA-DEPENDENT ATPASE ACTIVITY 
AND THOUGHT TO HAVE DIFFERENT 
CELLS IN THESE DIFFERENT STEPS 
OF EITHER TRANSCRIPTION, IE 
SERVELY, TRANSLATION, TURNOVER 
OF RNA AS EITHER RNA 
[INDISCERNIBLE] CASES OR AS RNP 
REMODELLING ENZYMES SO THAT THEY
WOULD EITHER UNWIND RNA DUPLEXES
OR REMEMBER IN TERMS OF MY SLIDEZv
WHERE I TALKED ABOUT RNA BINDING
PROTEINS HAVING AN IMPACT ON THE
FATE OF THE MEASUREN JER RNAs, 
IT WOULDYmOzBÑ EFFECTIVELY MODULATE 
WHICH RNA BINDING PROTEINS ARE 
BOUND TO A RNA AT DIFFERENT E 
STEP.
THAT'S WHAT WE REFER TO MRNP 
REMODELLING.
WHAT WE WERE ABLE TO DETERMINE 
BY FOCUSING ON THIS PARTICULAR 
DEAD BOX PROTEIN RIGHT HERE, 
IT'S A NUCLEAR PORE COMPLEX, TWR
FUNCTIONS FOR GLEE ONE AND IP 6 
AND mRNA EXPORT.
THIS DEAD BOX PROTEIN HAS BEEN 
IDENTIFIED IN MULTIPLE 
LABORATORIES AS HAVING ANç 
ESSENTIAL ROLE IN mRNA 
TRANSPORT.
THAT'S IN BOTH HUMAN AND IN 
YEAST CELLS.
SO WHAT WE SHOWED AND I'M GOING 
TO SHOW YOU A COUPLE OF PIECES 
OF DATA TO SUPPORT THIS, IS THAT
GLEE ONE DOCS AT THE NUCLEAR 
POREç COMPLEX.
JUST TO POSITION TO A DOCKING 
SITE FOR THIS DEAD BOX PROTEIN 
AND THAT THIS POSITIONING ALLOWS
ACTIVATION OF THIS DEAD BOX 
PROTEIN TO MEDIATE REMODELLING 
OF THE TRANSPORT RECEPTOR ON THE
MRNP AND OTHER SHUTTLING HMRNP 
PROTEINS THAT ARE BOUND TO THE 
mRNA.
IN TERMS OF THE EFFECT OF THE 
DEAD BOX PROTEIN, UM, THIS -- WE
WERE REALLY ABLE TO DOCUMENT 
HARD WORK AT ONE BEING ABLE TO 
PURIFY BOTH GLEE ONE AND DBP 5 
AND WHENEVER I GIVE THIS TALK 
 LIKE
AND SHOW THIS GEL, IT LO/%C:
IT WAS VERY STRAIGHTFORWARD, 
HOWEVER, THIS HAD BEEN A GOAL 
SINCE ROB MURPHY'S GAYDAY TO BE 
ABLE TO PURIFY GLEE ONE AND 
THERE WAS NO GIVING UP AND WAS 
ABLE TO IDENTIFY PURIFYING.
IP 6, THIS IS ACTUALLY IP 6 
WHICH YOU CAN BUY IN A VITAMIN 
STORE AND HE GOT THIS OFFER THE 
INTERNET TO PUT IT ON HERE, BUT 
THAT'S THE LEAST OF OURfá PROBLEMS
WHEN WE ARE RECONSTITUTING THIS 
SYSTEM IN VITRO.
WHAT WE WERE ABLE TO SHOW IS 
THAT DBP 5 ACTIVITY HYDROLYSIS 
OF AB FOSHGS ADP BY ITSELF HAS 
THIS LEVEL.
WHEN WE ADD BOTH GLEE ONE AND IP
6 THIS IS SIGNIFICANTLY 
STIMULATED.
IN PARTICULAR WHEN WE LOOK AT 
THE AMOUNT OF RNA THAT'S NEEDED 
TO STIMULATE THAT ATPASE 
ACTIVITY, THIS CURVE HERE IS THE
AMOUNT OF RNA ON A LOG SCALE 
THAT'S NEEDED WHEN DB FIVE IS BY
ITSELF BUT IT SHIFTS LOWER.
YOU NEED MUCH LESS RNA TO 
STIMULATE THE ATPS ACTIVITY IN 
PRESENCE OF GLEE ONE AND IP 6.
WHAT WE'VE BEEN ABLE TO SHOW 
MOST RECENT SLI THAT GLEE ONE 
ALSO DIRECTLY STIMULATES ATP 
LOADING ON TO DBP FIVE.
SIMILAR WORK IN TERMS OF THESE 
INITIAL CONCLUSIONS WAS ALSO 
REPORTED BY ANOTHER LABORATORY.
NOW, WHAT KRISTIN HAS BEEN ABLE 
TO GO ON TO DO WHICH WE FINDç 
VERY EXCITING IS THE FWLEE ONE 
STIMULATES THE ATP LOADING AND 
ATPASE ACTIVITY OF DBP FIVE AND 
THEN DBP FIVE IS BOUND TO ADP.
WHAT SHE HAS FOUND IS THAT 
ACTUALLY THE NUBS THAT ADP FIFE 
BINDS TO IS REQUIRED FOR RELEASE
OFçç THAT A ADP.
THIS IS AN EQUILIBRIUM BINDING 
WITH RADIO ACTIVE ADP AND FOR 
TEN MINUTES OR 24 HOURS 
AFTERWARDS IT STAYS VERY STABLY 
BOUND.
IF SHE ADDS GLEE ONE TO THIS, 
NOTHING HAPPENS, THE ADP STAYS 
DOWN.
IF HE ADD RNA, NOTHING HAPPENS, 
IT STAYS BOUND.
BUT IF SHE ADDS PURIFIED BINDING
SITES -- THIS IS THE DOUBENI IT 
BINDS TO HERE TAD SPSHGS 
RELEASED.
IF SHE USES MUTANTS IN JUNE 1, 
'59 THAT BLOCK THIS INTERACTION,
THEY DO NOT RELEASE THE ADP FROM
DBP FIVE.
IF YOU THINK OF THIS AS A CYCLE,
DTP FIVE IS AN ATPH AND IT'S 
CYCLE BETWEEN THE ADP AND 
ADP-BOUND STATE.
BOTH OF THESE IS A NUCLEAR CORE 
COMPLEX ARE FACILITATING THIS 
CYCLE WHERE IN GLEE ONE IS 
FACILITATING ADP LOADING AND NUB
159 IS FACILE DATING ADP 
RELEASE.
YOU COULD THINK OF THIS 
POTENTIALLY THAT THESE DEAD BOX 
PROTEINS HAVE BEING REGULATED IN
A VERY SIMILAR MATTER TO WHICH G
PROTEINS ARE REGULATED WHERE IN 
THEY'RE REGULATED BY EXCHANGEçó 
FACTORS AND I'M REFERRING TO AN 
ADP RELEASE FACTOR AND ALSO 
REGULATED BY GAS OR ACTIVATING 
FACTORS AND I'M REFER HERE TO 
GLEE ONE.
SO THINKING OF THIS AS A CYCLE 
WHICH IS HAPPENING AT THE 
NUCLEAR POREçç COMPLEX ON THE 
CYTOPLASMIC FACE, WE HAVE GLEE 
ONE STIMULATING ATP LOADING AND 
THE ATP ACTIVITY AND NUB 159 
STIMULATING ADP RELEASE AND 
COMING OFF WHEN THE ADP IS 
BOUND.
ALSO IT'S KIN TO G PROTEINS 
WHICH UNDERGO A NUKICALLY OWE 
TIDE CONFIRMATIONAL CHANGE 
STUDIES OF THE ADP-BOUND FORM OF
HUMAN DBP FIVE BY A EUROPEAN 
CONSORTIUM AND OF THE HUMAN DBP 
FIVE BOUND TO RNA BY ANOTHER 
LABORATORY ACTUALLY SHOWS, 
AGAIN, THIS çATP TO ADP 
CONFIRMATIONAL CHANGE.
WHAT'S ALSO INTERESTING IN THESE
STRUCTURES IS THAT THE RNA 
BINDING SITE HERE IN THE ADP 
BOUND FORM COMPARED TO WHAT 
WHERE IT WOULD HAVE BEEN IN THE 
SAME SIDE ON THE ADP BOUND FORM 
ALSO SIGNIFICANTLY CHANGES.
SO JUST THINGS THAT THE RNA 
WHICH IS BOUND CAN BE 
CONFORMATIONALLY CHANGED TO RI 
LOU REMODELLING OF ANY BOUND 
PROTEIN.
SO IN TERMS OF HOW ARE 
MACROMOLECULES TRANSPORTED 
THROUGH THE NUCLEAR PORE 
COMPLEX, WE HAVE FOUND THAT 
THERE IS A SET OF ESSENTIAL 
FACTORS ON THE CYTOPLASMIC OF 
THE CORE COMPLEX, SO THEY'RE 
REQUIRED TO EXECUTE A SPECIFIC 
ENZYME STEP ON THE MRNP BEING 
TRANCE LOCATED THROUGH.
WE SPECULATE THAT THISñr LOCALIZED
ACTIVATION OF THIS DEAD BOX 
PROTEIN ON THE CYTOPLASMIC FACE 
OF THE NUCLEAR PORE COMPLEX 
PROVIDES DIRECTIONAL CONTROL 
OVER THE EXPERT OF THATçó MRNP 
SUCH THAT IT DOES NOT COME OUT 
INTO THE CYTOPLASM AND GET 
REIMPORTED BACK INTO THE 
NUCLEUS.
IS A CRITICAL STEP IN TERMS OF 
REMOVING THOSE TRANSPORT FACTORS
AND REMOVING mRNA FINDING 
PROTEIN WHICH IS MIGHT HAVE 
INHIBITOR OR DETRIMENTAL 
FUNCTIONS WITHIN THE CYTOPLASM.ú
SO NOW FROM THIS STUDY OF THESE 
FACTORS, ARE THERE ANY 
POTENTIALLINGS TO CONNECTIONS 
BETWEEN EXPORT AND THE 
CYTOPLASMIC FATE OF THE myMRN 
SNSHGS ARE ANY OF THE ACTIONS 
WHICH ARE HAPPENING HERE 
IMPACTING TRANSLATION OR 
TURNOVER OR LOCALIZATION?
IN PARTICULAR, ANOTHER LAB ASKED
A QUESTION IN TERMS OF WHETHER 
OR NOT IS THERE A LINK 
BETWEEN -- IS GLEE ONE LANG 
BETWEEN mRNA EXPORT AND 
TRANSLATION.
I ALSO DESCRIBED TO YOU HOW 
HUMAN GLEE ONE HAS LOCALIZED 
MULTIIZATIONS THAT SUTING TWEEL 
THE NUCLEUS AND THE CYTOPLASM.
WE'D ALSO PREVIOUSLY FOUND THAT 
HUMAN GLEE ONE CAN INTERACT WITH
A SUB UNIT OF THE INITIATION 
FACTOR THREE.
THEN AROUND THIS SAME TIME, 
ANOTHER LABORATORY INñr GERMANY 
REPORTED THAT MUTANT ALLELES OF 
DBP FIVE THAT GLEE ONE ACTIVATE 
AT THE NUCLEAR CORE COMPLEX 
DURING mRNA EXPORT HAS A 
POTENTIAL ROLE IN TRANSLATION 
TERMINATION.
WE SPECULATE THAT BASED UPON 
THESE THIS EVIDENCE THAT GLEE 
ONE MIGHT ALSO HAVE A ROLE IN 
TRANSLATION REGULATION IN 
ADDITION TO AND SEPARATE FROM 
ITS ROLE IN mRNA EXPORT.
INDEED, TIM WAS ABLE TO USE A 
NUMBER OF DIFFERENT APPROACHES 
BOTH GENETIC AND BIOCHEMICAL TO 
DOCUMENT THAT GLEE ONE IP 6 DO 
PLAY A ROLE INxD TRANSLATION 
TERMINATION POTENTIALLY WE 
SPECULATELY ACTIVATING DBP FIVE 
IN THE SAME MANNER THAT IT 
ACTIVATES THE SAME DURING mRNA 
EXPORT.
IN ESSENCE, IF THIS IS ALLOWING 
SPATIAL CONTROL OVER REMODELLING
HERE, THE SPATIAL CONTROL OVER 
REMODELLING OF THE MRNP AT THIS 
FATE WOULD BE DICTATED 
SPECIFICALLY TO THIS TRANSLATION
TERMINATION STEP.
THIS ISS7 POTENTIALLY VIA 
INTERACTION WITH ERF ONE AND 
POTENTIALLY ALLOWS REMODELLING 
OF THIS TERMINATION COMPLEX SUCH
THAT ERF THREE OR SUB 35 CAN BE 
RECRUITED INTO THAT COMPLEX AND 
ALLOW SERUM NATION TO HAPPEN 
FAITHFULLY.
SO WE STILL HAVE SIGNIFICANT 
AMOUNT OF WORK TOç DO ON THIS 
PARTICULAR MECHANISM TO REALLY 
RESOLVE THAT IT INVOLVED AN MRNP
REMODELLING STEP IN THE SAME WAY
THIS INVOLVED MRNP REMODELLING.
WHAT TIM ALSO FOUND OUT IN THIS 
WORK IS THAT THE [INDISCERNIBLE]
ASSAYED FOR TRANSLATION EFFECT 
NOT ONLY HAD TRANSLATION 
TERMINATION DEFECTS BUT ALSO 
DEFECTS IN TRANSLATION 
INITIATION, AND THIS WAS 
POTENTIALLILINGED TO THIS 
INTERACTION WITH EIF THREE.
NOW, INTRIGUINGLY THE PRODUCTION
OF O IP 6 IN CELLS THAT LACKED 
PRODUCTION OF IP 6 DID NOT HAVE 
A DEFECT IN THE TRANSLATION 
INITIATION ASSAYS AND THE MUTANT
ALLELES IN DBP FIVE ALSO DID NOT
HAVE A DEFECT IN TRANSLATION 
INITIATION.
WE SPECULATED THAT THERE WAS A 
DIFFERENT DEAD BOX PROTEIN THAT 
WAS REQUIRED OR THAT GLEE ONE 
COULD BE AFFECTED DURING 
INITIATION AND GOING BACK TO 
THIS BLOWUP OF THIS PART OF THE 
DEAD BOX FAMILY PICTURE, THERE 
ARE MULTIPLE DIFFERENT DEAD BOX 
PROTEINS THAT ARE POTENTIALLY 
PLAYING ROLES IN TRANSLATION AT 
DIFFERENT STEPS.
AND SO IN SOME PRELIMINARY 
STUDIES, WE THINK WE HAVE FOUND 
A CONNECTION BETWEEN GLEE ONE 
REGULATING A DIFFERENT DEAD BOX 
PROTEIN, ONE CALLED DEAD ONE, 
WHICH OTHERS HAVE EXTENSIVELY 
CHARACTERIZED.
THIS IS AN ESSENTIAL DEAD BOX 
PROTEIN AND E POTENTIALLY PLAYS 
A ROLE IN SCANS BY ONE WINDING 
THE SECONDARY STRUCTURE.
WHAT TIM WAS ABLE TO FIND BY 
USING A GENETIC APPROACH TO 
START WITH IS THAT A DOUBLE 
MUTANT BETWEEN GLEE ONE AND DEAD
ONE SHOWED A SPECIFIC GENETIC 
INTERACTION A.
SO THIS PARTICULAR DEAD ONE 
MUTANT IS A COLD-SENSITIVE 
MUTANT WHERE IRN IT'S DEAD AT 25
A DEGREES AND ALIVE AT ALL OTHER
TEMPERATURES.
GLEE ONE MUTANT IS DEAD AT 37 
AND VERY, VERY SICK AT 30 AND A 
LIVE AT THE OTHER TEMPERATURES.
BUT THIS DOUBLE MUTANT SHOWED 
SPECIFIC SUPPRESSION OF THIS 
GLEE ONE TEMPERATURE SENSITIVE 
DEFECT AT 30 DEGREES.
AND IN ONE SET OF FOLLOW-UP 
EXPERIMENTS, WE POTENTIALLY 
THINK THAT GLEE ONE PLAYS A ROLE
IN INHIBITING THE ATPASE 
ACTIVITY OF DEAD ONE.
THIS SHOWS YOU THE EFFECT OF 
FWLEE ONE ON ACTIVITIES THAT I 
ALREADY DESCRIBED TO YOU THAT IT
ACTIVATES IT.
FOR DEAD ONE WITH PURIFIED 
REKOFSH INNOCENT DEAD ONE AND 
GLEE ONE WE DO NOT SEE 
ACTIVATION BUT RATHER INHIBITION
OF ACTIVITY.
IP 6 HAS NO EFFECT ON THIS 
INHIBITION WHICH CORRELATES WITH
THE IN VIVO RESULTS THAT IP 6 
HAS NO -- THE LACK OF IT HAS NO 
EFFECT ON IN VIVO TRANSLATION 
INITIATIONx÷ASSAYS.
SO TAKING ALL OF THESE STEPS 
TOGETHER WE SPECULATE THAT GLEE 
ONE TARGETED MULTIPLE DEAD BOX 
PROTEINS TO MODULATE GENE 
EXPRESSION BOTH TURN mRNA 
EXPORT, TRANSLATION INITIATION 
AND TERMINATION, AND THEN IT 
DOES THIS POTENTIALLY 
DIFFERENTIALLYç ACTIVATING ONE 
DEAD BOX PROTEIN AND INHIBITING 
ANOTHER ONE.
BUT THIS IS EARLY DAYS IN TERMS 
OF OUR ANALYZING THIS PARTICULARd8ç
MECHANISM AND WE HOPE TO BE ABLE
TO UNRAVEL THAT FURTHER.
WHAT I'VE DESCRIBED TO YOU SO 
FAR IS REALLY OUR STUDYING THE 
ROLE OF ONE PARTICULAR FACTOR IN
NUCLEAR TRANSPORT; HOW IT WORKS 
IN A NORMAL CELL AND WHAT IT IS 
INTERACTING WITH IN TERMS OF 
INTERACTION PARTNERS.
GOING BACK TO MY INITIAL PREMISE
IN TERM OF THE FACT THAT THIS 
MIGHT HAVE AN IMPACT IN STUDYING
DISEASE MECHANISM F YOU WERE TO 
HAVE ASKED ME WHAT DISEASE WOULD
I THINK GLEE ONE WOULD IMPACT I 
PROBABLY WOULD NEVER HAVE 
GUESSED THE ONE THAT A SET OF 
ENGLISH WORKERS LINKED IT TO AND
THAT IS A DISEASE CALLED LCCS 
ONE.
WHAT I'M GOING TO DESCRIBE TO 
YOU FOR THE LAST PART OF MY TALK
IS HOW WE'RE REALLY USING OUR 
INSIGHTS FROM STUDYING GLEE ONE 
AND YEAST IN HUMAN CELLS TO TRY 
TO UNROVL HOW IT HAS HAVING AN 
EFFECT ON N THIS LETHAL MOTOR 
NEURON DISEASE.ç
SO LCCS ONE IS AN AUTOSOMAL 
RECEPTIVE DISORDER.
1% OF THE POPULATION IS Av: 
CARRIER.
HAS BEEN IMPLICATED AS 
DEGENERATION OF THE MOTOR 
NEURONS AS THE PRIMARY CAUSE.
VERY INTERESTINGLY, AND THIS YOU
CAN USE TO THINK ABOUT AS YOU 
REFLECT ON THIS LATER.
THERE ARE TWO OTHER REPORTED 
LCCS ONE DISEASES, OTHER FORMS 
OF IT, TWO AND THREE, AND BOTH 
OF THESE HAVE BEEN 
GENETICALLILINGED TO ENZYMED 
INVOLVED IN FOS FI TITLE 
PATHWAYS.
[INDISCERNIBLE].
NEITHER OF THESE ARE 
DIRECTLILINGED TO IP 6 
PRODUCTION, BUT IT'S QUITE 
POSSIBLE THAT PERTURBING THEM 
MAY IMPACT THE SOLUBLEç POLLY 
PHOSPHATE POOLS AS WELL AS THE 
PATHWAYS, AND THE FACT THAT 
THESE ARE LINKED, I THINK, WILL 
BE A PRIME FOCUS OF OUR FUTURE 
STUDIES.
IN TERMS OF LCCS ONE, THE HOMO 
ZYGOTE MUTANT WHICH RESULTS IN 
THIS PHENOTYPE HAS BEEN MAPPED 
TO AN INCORRECT SPLICING DUE TO 
AN INCORRECT SLICING MUTATION 
THAT RESULTS IN A THREE AMINO 
ACID INSERTION IN THIS DOMAIN OF
GLEE ONE.
THE C TERMINAL DOMAIN OF GLEE 
ONE IS WHAT'S NECESSARY AND 
SUFFICIENT FOR ACTIVATING THE 
DEAD BOX PROTEIN AND FOR BINDING
IP 6.
THIS PARTICULAR DOMAIN OF GLEE 
ONE RIGHT HERE, UM, YOU'LL 
NOTICE IT SAYS POSSIBLE PROTEIN,
PROTEIN INTERACTION PARTNERS.
THROUGHyM ALL THE TWO HYBRID 
SCREENS WE'VE DONE OVER THE 
YEARS, WE HAVE NOT YET 
IDENTIFIED A PROTEIN INTERACTION
PARTNER FOR THIS DOMAIN, 
ALTHOUGH ANOTHER LAB HAS A PRIME
CANDIDATE THAT WE'LL BE 
FOLLOWING UP ON.
I'M GOING TO TALK TO YOU ABOUT 
THE WORK OF LEANNE WHO IS A POST
DOC FELLOW IN THE LAB WHO IS 
DOING A SECOND POST DOC WITH ME.
HE DID THE FIRST ONE HERE AT THE
NIH.
WHAT LEANNE HASç CHARACTERIZED IS
A SET OF ZEBRAFISH GLEE ONE 
MUTANT AND ZEBRAFISH KNOCKDOWNS 
OF GLEE ONE.
WHAT HE FOUND WITH THIS 
PARTICULAR MUTANT IS THAT THESE 
GLEE ONE MUTANTS HAVE LCCS 
ONE-LIKE S7PHENOTYPES.
THIS IS GOING TO ENABLE US TO 
MAP WHAT THE S THE PRIMARY 
PHYSIOLOGICAL DEFECT WHEN YOU 
HAVE DEFECTIVE GLEE ONE IN A A 
DEVELOPING VERTEBRAE SYSTEM.
SO THEY HAVE A CURVED BODY.
THEY HAVE IMMOW TALL.
THEY HAVE SUR FIVED TO THIS 
ñ 
LEVEL OF DEVELOPMENT DUE TO THE
MATERNAL PULL OF GLEE ONE.
IF YOU DO HE-STAINED PLASTIC 
SECTION, THIS IS THE WILD TYPE 
AND THIS IS THE SPINAL CORD 
HERE.
YOU CAN SEE IN THE GLEE ONE 
MUTANT THERE'S A THINNER SPINAL 
CORD.
THERE'S ALSO EDEMA AND 
UNDERDEVELOPED GUT SYSTEM, BOTH 
ALL OF WHICH PARALLEL THEOK 
REPORTED PATHOLOGY OF THE HUMAN 
DISEASE.
AND ALSO, THERE IS SEVERE PERTi] 
BASES IN CRANIOFACIAL 
DEVELOPMENT.
THIS IS STAINING OF THE CART 
LEDGE IN THE HEAD OF A WILD TYPE
FISH COMPARED TO THIS GLEE ONE 
KNOCKOUT.
THIS IS FROM A REVIEW SHOWING 
YOU THAT THE NORM -- WHAT THE 
NORMAL LOOKS LIKE.
SO IN THIS GLEE ONE KNOCKOUT, 
THE NEUROCRANIUM WAS MOSTLY 
NORMAL BUT THE CART LEDGES WERE 
NEARLY COMPLETELY ABSENT.
AGAIN, THIS CORRELATES WITH THE 
PATHOLOGY IN THE HUMAN DISEASE.
NOW IN TERMS OF MAPPING AND 
LOOKING DIRECTLY AT THE MOTOR 
NEURONS, WHAT LEANNE HAS BEEN 
ABLE TO DO BY IMMUNOSTAINING 
DIFFERENT POPULATIONS OF MOTOR 
NEURONS WITHIN THESE MU UH TANT 
AND KNOCKDOWN ZEBRAFISH IS FIND 
THAT THE MOTOR NEURON POOL IS 
SPECIFICALLY DEPLETED.
IF YOU COMPARE TO SENSORY 
NEURONS THERE'S NO DIFFERENCE 
BETWEEN WILD TYPE AND MUTANT, 
BUT WITH THE GLEE ONE KNOCKDOWN,
THERE'S ABOUT 25% FEWER MOTOR 
NEURONS.
AND MORE SO BECAUSE OF THE 
ELEGANT MICROSCOPY OFFERED BY 
THE ZEBRAFISH SYSTEM, HE CAN 
LOOK DIRECTLY AT THE DEVELOPMENT
OF THOSE MOTOR NEURONS AND AT 
THE ARBOR RYIZATION.
LOOKING AT WILD TYPE HERE URIC 
CAN SEE LABELING OF THE MOTOR 
NEURONS IN RED BODIES AND THEN 
ALONG HERE YOU HAVE THE MOTOR 
NEURONS AND EXXONS EXTENDING 
FROM THE WILD TYPE IN THIS VERY 
CLASSIC STRUCTURE.
IN THE GLEE ONE MUTANTS YOU SEE 
FEWER MOTOR NEURONS ALONG THE 
TOP HERE BUT THEN MORE SO THESEq
EXXONS HAVE ASH RYIZATION 
DEFECTS IN TERMS OF THE AXONS 
THAT ARE COMING OFF OF THEM.
ONE OF THE FIRST EXPERIMENTS WE 
TRIED TO DO IS TO SEE WHETHER OR
NOT WE CAN COMPLIMENT THIS 
PHENOTYPE TO MAKE SURE IT'S THE 
DIRECT PHENOTYPE FROM THE 
KNOCKOUT OR IF WE'RE LOOKING 
FROM THE KNOCKDOWN OF GLEE ONE.
IN PARTICULAR, WE HAVE DONE THIS
BY EXPRESSING THE HUMAN FORM OF 
GLEE ONE IN THE ZEBRAFISH AND WE
GET REMARKABLE COMP MENATION AND
RESCUE WITH THE WILD TYPE.
THIS SHOWS YOU WITH THE MOFALINO
EXPERIMENT HOW WE CAN GET Amy 
DEFECT, AND HERE BY EXPRESSING 
THE WILD TYPE HUMAN FORM OF GLEE
ONE IN THESE FISH THAT HAVE THE 
ZEBRAFISH KNOCKED OUT, WE GET 
RESCUE OF THE PHENOTYPE.
IF WE EXPRESS INSTEAD THE THIN 
MAJOR VERSIONLINGED TO THE 
DISEASE, WE DO NOT GET RESCUE OF
THE DEFECT.
THEREFORE, WE THINK THAT THIS 
WILL BE A PRIMARY AND A VERY 
GOOD SYSTEM FOR TESTING THE 
PHENOTYPES OF THOSE DISEASE 
MUTANTS.
NOW NEWEST WORK THAT LEANNE HAS 
DONE IS TO REALLY TRY TO 
PINPOINT WITHIN THE 
DEVELOPMENTAL PATHWAY OF THOSE 
MOTOR NEURONS CHA WHAT IS THE 
PRIMARY DEFECT?
AND SO TO DO THIS, AGAIN, WE CAN
TAKE ADVANTAGE OF THE POWER OF 
THE ZEBRAFISH SYSTEM AND REALLY 
DIFFERENTIALLY LABEL DIFFERENT 
CELL POPULATIONS AND MONITOR 
THEM THROUGHOUT THE TIME COURSE 
OF DEVELOPMENT.
AND SO O IN THIS CASE, THE 
QUESTION WAS TRYING TO DETERMINE
WHETHER OR NOT THE FEWER NUMBERS
OF MOTOR NEURONS THAT EXISTED 
WAS DUE TO THOSE MOTOR NEURONS 
DYING OR WAS IT DUE TO NEURAL 
PROGENITORS DYING?
SO THIS IS A CARTOON DIAGRAM OF 
ESSENTIALLY ONE HALF OF THIS ç
IMAGE.
THIS IS A SLICING THE FISH DOWN 
THE MIDDLE SO THAT YOU HAVE A 
FEW OF THE ENTIRE SPINAL AREA 
RIGHT HERE, AND THIS IS WHERE 
THE CANAL IS ALONG HERE, AND SO 
ALONG THIS THE DEVELOPMENTAL 
PATHWAY, THE PROGENITORS START 
OUT RIGHT ALONG THIS LINE HERE.
OKAY.
AND THEN AS TIME PROGRESSES, 
THEY DIFFERENTIATE AND MIGRATE 
TOWARDS THE OUTSIDE EDGE, AND AS
THEY DIFFERENTIATE, THEY BECOME 
LABELED BY DIFFERENTIATION WITH 
THIS PARTICULAR MARKER, AND IN 
THIS CASE THAT'S SHOWNç IN RED.
THAT'S WHY THE DIFFERENTIATED 
MOTOR NEURONS ARE IN RED AND 
THEN ALONG THE MIDDLE WE HAVE 
THE RADIO FWLEEL OR 
NEUROPROGENITORS IN GREEN.
SO IF WE LOOK AT THIS THIS, IS 
JUST IN A WILD TYPE SITUATION.
IF WE LOOK AT THIS AND THE 
MUTANT SITUATION IT ALSO 
COINCIDENTLY LABELED THE CELLS 
WITH CASPASE THREE WHICH MARKED 
CELLS CHAR UNDERGOING APOPTOSIS,
WE CAN MAKE THE CONCLUSION THAT 
IT'S THE NEUROPROGENITORS THAT 
ARE SELECTIVELY DIEING WITH THE 
LOSS OF GLEE ONE.
AGAIN, HERE'S IN PURPLE ARE THE 
CASPASE THREE APOPTOSISñr CELLS 
AND THEY ARE ONLY COLABELED WITH
GREEN, WHICH IS REFLECTING THE 
NEUROPROGENITORS.
WE NEVER SEE COLABELING BETWEEN 
A CASPASE AND BETWEEN THE RED OR
THE ALREADY-COMMITTED, DEVELOPED
MOTOR NEURONS.
SO IN TERMS OF SPECULATING ON 
THE MOLECULAR DEFECT IN THIS 
HUMAN GLEE ONE SIMILAR MUTATION,
AT THIS POINT BASED UPON THEç 
RESULTS WE HAVE IN ZEBRAFISH, I 
WOULD CROSS OUT A SPECIFIC 
DEFECT IN ANY OF THESE 
PARTICULAR STEPS.
AND IF YOU HEARD MY TALK, YOU 
KNOW, TWOw3d8 MONTHS AGO OR SO AT 
ANOTHER CON PROTECT THE, I WOULD
HAVE PROBABLY IMPLICATED 
TRANSLATION AND LOCALIZATION 
THAT GLEE ONE WAS AFFECTING ONE 
OF THOSE FUNCTIONS.ç
BUT WHAT WE ACTUALLY THINK RIGHT
NOW THAT HIGHLY MRIF RATING 
CELLS, FOR EXAMPLE THESE 
NEUROPROGENITORS, ARE 
SPECIFICALLY AFFECT BID THE 
THRESHOLD LEVEL POTENTIALLY OF 
EVERY SINGLE STEP ALONG THIS 
PATHWAY THAT GLEE ONE IS 
IMPACTING.
THEREFORE, THAT'S WHY THE KNEE E
KNOW TYPE IS MANIFESTED INçó THOSE
PARTICULAR HIGHLY PROLIFERATING 
CELLS.
SO THAT ALSO COULD SHOW THAT 
POTENTIALLY FACTORS WHICH ARE 
MULTI FUNCTIONALLY LINKING 
MULTIPLE STEPS ALONS THIS GENE 
EXPRESSION PATHWAY MIGHT BE MORE
SUSCEPTIBLE TARGETS THAN THESE 
TYPES OF DISEASES BECAUSE THEY 
WOULD BE HIGHLY REQUIRED BY 
HIGHLY MRIF RATING CELL TYPES.
SO THE TYPES OF FACTORS WHICH WE
HAVE BEEN ABLE TO LINK TOGETHER 
INCLUDE THE DEAD BOX PROTEINS, 
GLEE ONE, AND ALSO THE 
REQUIREMENT OF THESE POLLY 
PHOSPHATES.
SO IF I TAKE YOU THROUGH THIS 
WHOLE TIME COURSE, I REALLY 
THINK THAT THESEd8 ARE UNEXPECTED 
DISCOVERY BECAUSE I WOULDN'T 
HAVE PREDICTED GOING INTO A 
ZEBRAFISH MODEL FROM STUDYING 
mRNA EXPORT FACTORS AND TRACKING
DOWN THEç PROLIFERATION AND 
GROWTH ACTIVITY OF 
NEUROPROGENITORS.
THAT'S WHAT MAKES DOING SCIENCE 
SO VERY EXCITING.
I THINK THE OTHER O ASPECT OF 
THIS THAT I HAVEN'T TOLD YOU 
ABOUT IS THAT AL A THOUGH IN THE
PARTICULAR CASE OF GLEE ONE 
WE'VE LINKED IT TO A THRESHHOLD 
STEP IN HIGHLY PROLIFERATING 
CELLS WE'VE GOTTEN OTHERçó%q 
POTENTIAL DISEASELING WHICH ARE 
MORE DIRECT AND MEK NISTY FROM 
STUDYING SPECIFICALLY THE 
PRODUCTION OF THOSE SOLUBLE 
POLLY PHOSPHATES.
AGAIN, USING THE ZEBRAFISH 
MODEL.jF
ALTHOUGH I DON'T HAVE TIME TO 
TELL YOU ABOUT THESE, UM, ONE 
WAS JUST PUBLISHED A COUPLE OF 
MONTHS AGO AND THE OTHER ONE 
PUBLISHED OVER THE LAST COUPLE 
OF YEARS.
THESE ARE VERY INTRIGUING 
EXAMPLES WHERE IN THIS POLLY 
PHOSPHATES PATHWAY MAY BE 
SPECIFICALLY AFFECTING 
INVERTEBRATE CELLS MORE THAN 
JUST GLEE ONE.
SO I SHOWED YOU IN THAT ORIGINAL
DIAGRAM THAT THERE WERE MULTIPLE
FUNCTIONS THAT HAD BEEN LINKED 
TO THEM.
ONE THAT WE'VEw3 PLACED THEM TO IS
INSILL RI FUNCTIONS.
IN IP 6 IS NOT PRODUCED, SILL 
YEAH DO NOT BEAT.
IF YOU DON'T PRODUCE ONE OF 
THESE PYROPHOSPHATES, HEDGEHOG 
SIGNALING IS DIRECTLY IMPACTED.
IN TERMS OF FUTURE CHALLENGES, 
I'D SAY THAT OVER THE COURSE OF 
THE YEARS I'VE DISCOVERED A LOT 
OF PARTS AND MUCH OF THE 
MACHINERY THAT IS INVOLVED IN 
NEWICALLY OWE SKRIE TOE PLASMIC 
TRANSPORT HAS BEEN UNRAVELLED BY
PEOPLE WORKING IN THE FIELD AND 
WE CAN DIRECTLY ANALYZE THEM IN 
VIVO, BUT REALLY OUR CHALLENGE 
IS NOW TO BE ABLE TO STUDY THEM 
SPATIALLY, TEMPORALLY AND AS 
PARTS OF LARGER COMPLEXES AND 
REALLY INTERCONNECTED PATHWAYS 
ESPECIALLY WHENñr THEY'RE 
MULTIFUNCTIONAL.
I AM ESPECIALLY IN DEBT TO THE 
PEOPLE IN MY LABORATORY OVER ALL
THE YEARS.
THIS IS MY CURRENT GROUP AND 
THOSE WHO ARE IN RED ARE THE 
PEOPLE CURRENTLY IN THE LAB 
WHERE I DISCUSS THEIR 
UNPUBLISHED WORK TODAY.
THOSE HERE HAVE RECENTLY LEFT 
THE LAB FOR BIGGER AND BETTER 
POSITIONS THAT MADE CRITICAL 
CONTRIBUTIONS, AND, UM, BRUCE 
APPLE HAS BEEN A REALLY 
IMPORTANT COLLABORATOR IN ALL OF
OUR ZEBRAFISH WORK OVER THE 
YEARS AND RECENTLY COLLABORATING
WITH CHUCK COAL IN TERMS OF 
UNRAVELLING THE DTP FIVE ONE 
CYCLE AS THE NUCLEAR CORE t(
COMPLEX.
I'VE BEEN FUNDED BY GM SINCE THE
BEGINNING OF MY LAB AND I'M 
GRATEFUL FOR THAT AND WE HAVE 
NEW FUNDING FROM THE MARCH OF 
DIME AND AMERICAN HEART 
ASSOCIATION FOR SOME OF OUR 
ZEBRAFISH WORK AND I'M HAPPY TO 
TAKE QUESTIONS.
THANK YOU.
[APPLAUSE]
>> YOU HAVE THESE IP 6 GLEE ONE 
INTERACTING WITH DIFFERENT DEAD 
BOX PROTEINS DO YOU HAVE ANY 
SENSE OF HOW THEY'RE INTERACTING
WITH THE DIFFERENT PROTEINS AND 
WHETHER THAT CAN GIVE YOU 
INSIGHT AS TO WHY YOU HAVE ONE 
THAT'S ACTIVATED AND ONE THAT'S 
INHIBITED?
>> RIGHT.
SO THE DEAD BOX ARE THE DBP FIVE
STRUCTURES VSH OUT SINCE 2009.
CARSON JUST PUBLISHED LIKE TWO 
WEEKS AGO A STRUCTURE THE C 
TERMINAL REGION OF GLEE ONE WITH
DBP FIVE.
THE C TERMINAL REGION OF GLEE 
ONE LOOKS LIKE EIF 4 G AND 
THEREFORE THERE'S POTENTIAL 
ANALOGIES BETWEEN HOW DIFFERENTñr 
EFFECTORS COULD BE AFFECTING 
DIFFERENT DEAD BOX PROTEINS BUT 
IT'S REALLY EARLY DAYS IN TERMS 
OF ANALYZING THAT STRUCTURE.
ALSO OF NOTE THAT THE STRUCTUREfá 
PUBLISHED IS NOT OF WILD TYPE 
PROTEINS.
THIS IS A CREATIVE TRICK THAT 
THEY USED BECAUSE THE WILD TYPE 
PROTEINS WILL NOT CRYSTALLIZE 
TOGETHER AND SO THEY USED TWO 
DOMINANT ALLELES THAT HAVE A 
TIGHTER AFFINITY INTERACTION 
WITH ONE ANOTHER TO GET THAT 
CRYSTAL.
AND SO, UM, IN TERMS OF DEAD 
ONE, THERE IS NO CRYSTAL 
STRUCTURE OF THAT AND IF YOU TRY
TO LOOK FOR HOE SDWROI IDENTIFY 
WHAT COULD BE THE INTERACTION 
INTERFACE, WE HAVExD INTERACTION 
BETWEEN FWLEE ONE AND 
[INDISCERNIBLE] BUT I THINK IT'S
GOING TO TAKE ONE STRUCTURAL 
ANALYSIS AND TWO WE'RE STARTING 
WORK WITH THE ADP RELEASE AND 
THOSE TYPE OF ANALOGIES TO SEE 
IF THERE'S XHONLTS IN TERMS OF 
THAT.
COMMONALITIES IN TERMS OF THAT.
>> [LOW AUDIO].
>> THE IP 6 PHENOTYPES ARE -- SO
FIRST, IF YOU KNOCK DOWN IP 6 IN
ZEBRAFISH YOU ALSO GET DEFECTS 
IN MOTOR NEURON AND DEVELOPMENT.
I DIDN'T SHOW THAT, BUT YOU DO 
NOT GET DEFECTS IN CRANIAL 
FACIAL DEVELOPMENT IF YOU KNOCK 
DOWN IP 6.
SO THERE -- WE HAVE AN ASSAY 
[INDISCERNIBLE] FUNCTION IN GLEE
ONE KNOCKDOWNS BUT IP 6 IS GOING
TO HAVE MANY, MANY DIFFERENT 
TARGETS IN THE CELL.
WHEN YOU KNOCK DOWN IP 6 YOU'RE 
IMPACTING ALL THE DIFFERENT 
TARGETS THAT IT'S HAD WHICH 
INCLUDES ADAR, THE RNA EDITING 
ENZYME, GLEE ONE, POTENTIALLY 
COME SIGNALING RECEPTORS ON THE 
PLASMA MEMBRANE.
SO THAT'S WHY THEIR PHENOTYPES 
IN VERTEBRA ARE NOT GOING TO BE 
COMPLETELY OVERLAPPING.
NEW CELLSt( WHICH MIGHT BE SIMPLER
IN TERMS OF THE NUMBER OF 
TARGETS.
>> I'VE ALWAYS FIND 
[INDISCERNIBLE] FATE METABOLISM 
COMPLETELY PERPLEXING, EVEN IN 
SIMPLE ORGANISM LIKE YEAST, BUT 
I'M WONDERING IF YOU COULD AT 
LEAST SPECULATE WILDLY AS TO WHY
IP 6 IS BEING USED FOR THISç 
CRITICAL ROLE OF DOCKING.
>> THE PERPLEXING PART YOU DON'T
BRING UP IS THAT IT'S SUB CELLAR
LEVELS ARE VERY HIGH IN TERMS OF
VERY HIGH CONCENTRATIONS, 
ALTHOUGH WHAT IS THE PREPOOL 
VERSUS THE PROTEIN-BOUND POOL.
AkOsDASHGS DOES NOT FOLD WITHOUT 
IP 6 AND IP 6 IS LITERALLY 
EMBEDDED BEEN WITHIN THAT 
STRUCTURE.
FOR GLEE ONE, GLEE ONE FOLDS 
WITHOUT IP 6, WE CAN COMPLETE IP
6 OFF.
IT'S A VERY DIFFERENT TYPE OF 
INTERACTION THAN IT IS WITH A 
DAR.
JOHN YORK HAS PRESENTED RESULTS 
IN TERMS OF IDENTIFYING A WHOLE 
NEW UH FAMILY OF IP 6 BINDING 
PROTEINS.
I WOULD WILDLY SPECULATE THAT 
THERE ARE POTENTIALLY TARGETS 
WHICH ARE MODULATED AND 
POTENTIALLY TARGETS WHICH ARE 
USING IT MORE AS A FOLDING 
COFACTOR, AND THERE ARE THOSE 
TWO DIFFERENT TYPES, AND NOW 
IT'S GOING TO BE VERY DIFFICULT 
TO TEASE APART WHICH IS WHICH.
NOW, THE GLEE ONE DEAD ONE EVEN 
THOUGH IT DOESN'T REQUIRE IP 6 
AND IP 6 HAS NO EFFECT, IP 6 CAN
STILL BIND TO IT.
SO DEAD ONE DOESN'T PROHIBIT THE
BINDING OF IP 6 TO GLEE ONE.
MY SPECULATION IS THAT THERE'S 
GOING TO BE TWO DIFFERENT TYPES 
OF POOLS OF IP SIX.
>> ARE THERE ANY SPECIFIC 
INTERACTIONS BETWEEN GLEE ONE 
AND [INDISCERNIBLE]?
>> YES.
SO THERE ARE TWO DIFFERENT 
DOCUMENTED GLEE ONE/NUCLEAR 
[INDISCERNIBLE] INTERACTION.
ONE IS WITH THE CRY TO PLASMIC 
FACE NOOK.P,Ñ
THE OTHER HAS BEEN DOCUMENTED 
WITH HUMAN GLEE ONE WE HAVE 
FOUND AN INTERACTION WITH A NUB 
CALLED 155 WHICH IS LINKED TO 
HUMAN DISEASE WITH PATIENTS WITH
ATROE FIBRILLATION PROBLEMS.
THAT'S INTERESTING WHETHER OR 
NOT THE GROUPS WORKING ON THAT 
HAVE BEEN POTENTIALLY IMPLICATED
GLEE ONE TO THAT TOO.
>> AS A FOLLOW-UP QUESTION, YOU 
ENVISION THE ROLE OF GLEE ONE 
AND TRANSLATION AS SOMEHOW BEING
PART OF AN HMRP OR LOADING ON TO
THIS RMP WHEN IT'S LOADING ON TO
THE PORE OR INTERACTING WITH 
RIBOSOMES?
>> I DO NOT THINK IT GETS 
INCORPORATED INTO THAT MRNP AS 
ITS BEING xDEXPORTED BUT RATHER 
IT'S REDRUTED TO DIFFERENT 
[INDISCERNIBLE] EITHER IN EXPORT
OR TRANSLATION INITIATION OR 
TRANSLATION TERMINATION THAT 
EJECT éHVu! POSITION NEXT TO THE 
DEAD BOX PROTEIN IT'S GOING TO 
ACTIVATE.
ONE REASON WHY I DON'T THINK 
IT'S INCORPORATED IS BASED UPON 
THE CELLAR COPY NUMBER AND 
LEVEL.
DBP FIVE IS 20,000 COPIES 
ESTIMATED IN A YEAST CELL VERSUS
GLEE ONE TWO THOUSAND.
THERE'S NOT ONE FWLEE ONE FOR 
EVERY MESSENGER RNA OR 
TRANSLATION INITIATION OR 
TERMINATION.
IT HAS TO BE VERY RAPIDLY 
TURNING OVER AND CYCLINGñrç BETWEEN
THESE DIFFERENT FUNCTIONS.
>> I WANTED TO ASK YOU A 
QUESTION ABOUT THE ROLE OF THE 
NUB 159 IN RELEASING THE ADP.
FWLEE ONE AND DBP FIVE ARE ALSO 
WORKING AT TERMINATION, SO WHAT 
DO YOU THINK IS DOING THE ADP 
RELEASE -- I ASSUME NUB 159 -- 
>> NUB 159 A MUTANTS DO NOT HAVE
ANYxD TRANSLATION INITIATION.
IT'S KIND OF LIKE THE GOLD 
STANDARD CONTROL BY BOTH MY 
LABORATORY AND OTHER 
LABORATORIES FOR GLEE ONE AND 
DBP FIVE IN TERMS]I OF A NUB THAT 
DOESN'T HAVE A DEFECT.
WE SPECULATE THAT THERE IS 
ANOTHER FACTOR THAT CAN MEDIATE 
ADP RELEASE DURING TRANSLATION 
TERMINATION,u! AND POTENTIALLY 
THAT THAT'S HELPING RECYCLE DBP 
FIVE WHEN NUB 159 WHEN THAT 
BINDING IS ABSENT, CELLS ARE NOT
DEAD BUT THEY'RE TEMPERATURE 
SENSITIVE AND YOU CAN SUPPRESS 
THAT BY OVEREXPRESSING DBP FIVE 
AND POTENTIALLY IT'S A 
TRANSLATION TERMINATION 
TRIGGERING RELEASE FACTOR.
THAT COULD UH POTENTIALLY BE A 
PART OF THE RIBOSOME.
IT COULD BE SUBMY 45 OR 35.
THOSE ARE THE CANDIDATES THAT 
WE'RE GOING TO BEGIN TESTING 
DIRECTLY IN VITRO, BUT THEN IN 
INVIN VOE WE'RE GOING TO TRY TO 
USE THAT THAT LACKED THE DBP 
FIVE THAT DOESN'T BIND IT TO 
LOOK FOR MUTANTS WHICH ARE NOW 
DEAD WITHOUT THAT PUNITIVE 
TRANSLATION ADP RELEASE FACTOR.
>> [LOW AUDIO].
[APPLAUSE]fá
>> THANK YOU.
>> THANK YOU.