PDF file: EURASNET Annual Report 2008

Participant 1B – Karla Neugebauera) Work performed during the periodWP11: CBP20 and CBP80 together comprise the cap-binding complex (CBC), which promotes spliceosomeassembly. We discovered the expression of an alternatively spliced form of CBP20, called CBP20S, which lacksthe RNA recognition motif. Thus, CBP20S has the potential to be a negative regulator of spliceosome assembly.CBP20S was shown to be expressed by all primary and immortalized human cells tested. Although it does not bindCBP80, pre-snRNAs or 7-methyl-G sepharose, it does associate weakly with mRNAs and localizes to transcriptionunits in vivo. A predoc, Marta Pabis, worked on this full-time during 2008. A manuscript is in preparation,pending a few additional experiments to determine CBP20 binding partners.WP13:Our lab established splicing factor ChIP in yeast and mammalian cells; this includes the entirely novel strategy ofChIP of alternative splicing factors, which are expressed as tagged versions from stably transfected BACs. Theproof-of-principle paper, including an analysis of the dynamic recruitment of SR proteins has been provisionallyaccepted at Molecular Cell. The manuscript includes our analysis of differential splicing factor recruitment to theSAT III locus (with Biamonti). Use of BAC tagging has led to improvements over the use of antibodies andstimulated a number of new collaborations within the workpackage (Aubeouf, Bertrand, and Smith). Threeadditional projects were a focus in 2008: 1) Extension of the BAC-tagging scheme to P19 cells, which can bedifferentiated into neurons (postdoc Minni Änkö, predoc Lucia Morales); neuronal targets of SRp20 and SRp75have been identified. 2) Launching of the “model genes” project (predoc Nicole Bardehle), in which model genesfor co-transcriptional spliceosome assembly in situations of conditional or alternative splicing are created inmammalian cells. 3) Initiation of a “model genes” project in yeast (predoc Fernando Carillo-Oesterreich), in whichintegrated or plasmid-borne copies of co-transcriptional splicing reporters are analyzed to assess the contributionof chromatin to co-transcriptional splicing. The postdoc Ana Ruiz-Manzano, contributed to an aspect of this work,determining the role of TFIIS in transcription elongation and co-transcriptional splicing in yeast. Our collaborativeproject with Seraphin on the role of Pol II in U1 snRNP recruitment to nascent RNA is complete, with amanuscript nearly ready for submission.b) Major cost items with justificationParticipant 1B Type of expenditure Budget total costspersonnel 17.594,00consumables 18.688,33training 0equipment 0travel 0= total direct costs 36.282,33+ overhead 7.256,47= total eligible costs 43.538,80Major cost itemsResearchPersonnelMarta Pabis [WP 13] 1 PM (= 160 hours) 1,250 EuroFernando Carillo-Oesterreich [WP 13] 11 PM (= 1,760 hours) 14,850 EuroJanina Görnemann [WP 13] 1 PM (= 160 hours) 1,494 EuroConsumablesAntibodiesChip, Q-PCR reagentsChemicals + misc2,195.63 EUR4,331.47 EUR12,161.23 EUR131