title: The role of ZC3H32 in Trypanosoma brucei
creator: Klein, Cornelia Andrea
subject: 570
subject: 570 Life sciences
description: In  the  protozoan  parasite  Trypanosoma  brucei  gene  expression  is  mostly  regulated  on  the  post-­‐transcriptional  level.  C3H  zinc  fingers  have  shown  to  play  an  important  role  in  this  process.  So  far,  all  described  C3H  zinc  finger  in  Trypanosoma  brucei  have  shown  to  stabilize  their  targets.  This  work  focuses  on  the  zinc  finger  protein  ZC3H32.  A  large-­‐scale  isolation  of  free  and  membrane-­‐bound  polyribosomes  from  proyclic  and  bloodstream  trypanosomes,  which  is  also  described  in  this  thesis,  suggested  that  this  protein  associates  with  polyribosomes.  However,  small-­‐scale  polyribosome  isolations  followed  by  Western  blotting  revealed  that  only  a  minor  fraction  of  it  is  actually  associated  with  polyribosomes.  Immunofluorescence  showed  that  ZC3H32  localizes  to  the  cytoplasm.  A  yeast-­‐two-­‐  hybrid  screen  identified  it  as  a  putative  binding  partner  of  MKT1.  This  interaction  was  confirmed  by  co-­‐immunoprecipitation.  RNAi,  as  well  as  knock-­‐out  studies,  showed  that  ZC3H32  is  essential  in  the  bloodstream  form.  Northern  blotting,  as  well  as  a  SILAC  screen  by  Urbaniak  et  al.  indicate  that  it  is  also  enriched  in  this  life-­‐cycle  stage  as  compared  to  procyclics.  Artificial  tethering  of  ZC3H32  to  a  reporter  RNA  lead  to  the  RNA’s  degradation,  suggesting  that  this  protein  has  a  destabilizing  effect.  Tethering  of  ZC3H32  fragments  revealed  that  both  its  N-­‐terminal,  as  well  as  its  C-­‐terminal  region  are  able  to  generate  this  destabilization  effect,  while  the  middle  region,  containing  the  zinc  finger  domains,  can’t.  RNA  isolation  from  polyribosomal  fractions  showed  that  tethering  of  ZC3H32  also  decreases  the  translation  of  the  reporter  RNA.  High-­‐throughput  sequencing  of  poly-­‐A+  RNA  from  a  ZC3H32-­‐RNAi  cell  line  revealed  20  RNAs  that  were  up-­‐regulated  upon  ZC3H32  knock-­‐down  and  thus  might  be  putative  targets.  The  upregulation  was  confirmed  for  three  of  these  candidate  RNAs.  The  majority  of  the  putative  ZC3H32  targets  play  a  role  in  the  trypanosome’s  energy  metabolism  and  15  of  them  are  up-­‐regulated  in  procyclics.  These  results  suggest  that  ZC3H32  might  be  involved  in  the  stage-­‐specific  regulation  of  these  RNAs  in  the  bloodstream  form.
date: 2014
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/17057/1/Doktorarbeit%20Cornelia%20Klein.pdf
identifier: DOI:10.11588/heidok.00017057
identifier: urn:nbn:de:bsz:16-heidok-170572
identifier:   Klein, Cornelia Andrea  (2014) The role of ZC3H32 in Trypanosoma brucei.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/17057/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng