Gene  Regulation  Through  the  Biochemical  Production  of  MicroRNA  

By:  Karissa  Yamaguchi  
Edited  by:  Zhiye  Wang  
 
DNA  is  essentially  the  code  of  life.  Every  living  cell  contains  DNA  made  of  the  
same  nucleotide  building  blocks.  Genetic  information  is  crucial  to  the  formation  of  
life  and  is  inherited  through  each  generation.  The  central  dogma  of  genetics  is  that  
DNA,  the  genetic  code  within  all  cells,  is  transcribed  onto  messengerRNA,  which  
transports  that  genetic  information  to  be  translated  into  a  protein  product.  This  
simple  depiction  refers  to  DNA’s  expression  as  the  creation  of  proteins.  DNA  acts  as  
a  template  while  RNA  serves  as  a  messenger  of  the  information.  First,  the  
complementary  base  pair  structure  of  the  genetic  code  allows  for  the  creation  of  
RNA.  This  information  is  then  carried  by  the  messenger  RNA  to  a  ribosome  (a  large  
RNA-­‐Protein  molecular  machine),  which  then  translates  a  simple  string  of  bases  into  
an  amino  acid  and  eventually  a  protein  product.    
However,  only  a  small  percentage  of  the  human  genome  directly  encodes  for  
proteins  expressed  through  this  process.  Most  are  instead  responsible  for  the  
regulation  of  this  process.  My  research  under  Dr.  Zhiye  Wang  in  Dr.  Xiuren  Zhang’s  
lab  seeks  to  find  the  mechanism  for  this  regulation  by  a  specific  type  of  RNA—
microRNA.    
Past  research  has  revealed  how  miRNA  associates  with  proteins  and  
enzymes  to  form  a  tool  for  post-­‐translational  regulation.  Similar  to  the  transcription  
of  messenger  RNA  (mRNA),  microRNA  precursors  are  transcribed  from  the  DNA  
template.  After,  RNase-­‐Ⅲ    type  enzymes  (often  referred  to  as  Dorsha  and  Dicer  in  
humans  and  Dicer-­‐like  in  plants)  process  the  precursors  to  small  RNAs  such  as  
microRNA.  It  is  known  that  microRNA  is  then  incorporated  with  other  enzymes  into  
an  Argonaute-­‐centered  RNA-­‐induced  silencing  complex  or  RISC.  These  RISCs  are  
precise,  mRNA  cutting  tools,  thus  causing  the  degradation  of  mRNA  or  inhibiting  the  
translation  of  mRNA  to  amino  acids.  This  mechanism  effectively  reduces  the  

production  of  the  protein  produced  from  the  genes  transcribed  on  the  messenger  
RNA.  This  mechanism  thus,  silences  the  gene.  
Surprisingly,  though,  when  a  microRNA  precursor  (the  DNA  code  for  the  
production  of  microRNA)  is  inserted  after  a  gene  of  interest  (in  the  3’UTR),  the  
expression  of  that  gene  increases.  We  discovered  this  function  of  microRNA  by  
comparing  plants  genetically  engineered  with  the  same  bioluminescence  gene,  
differing  in  only  the  addition  of  a  microRNA  precursor.    
We  engineered  agrobacteria  with  a  ring  of  DNA  (a  plasmid)  including  a  
Transfer-­‐DNA  insert.  Some  of  these  Transfer-­‐DNA  inserts  contained  the  microRNA  
precursor  gene  in  addition  to  the  bioluminescence  gene—luciferase.    Luciferase  is  a  
reporter  gene.  Thus,  visualizing  the  plants  under  ultra-­‐sensitive  CCD  camera  
indicates  the  outcome  of  this  process.  A  successful  transformation  results  in  the  
formation  of  a  plant  that  glows.  After  infecting  plants  with  the  transforming  
agrobacteria,  the  plants,  which  had  received  the  miRNA  gene  in  addition  to  
luciferase,  expressed  a  greater  amount  of  luciferase  protein  through  more  glow  than  
those  only  receiving  luciferase.  
These  results  revealed  even  more  complexity  in  the  mechanisms  and  
pathway  of  microRNA  and  raised  several  unknowns.  Does  the  insertion  of  a  primary  
microRNA  after  a  gene  affects  the  transcriptional,  post-­‐transcriptional  or  
translational  level,  and  does  the  produced  miRNA  or  the  RNA  structure  affect  the  
regulation  of  that  inserted  gene?  
We  adopt  a  forward  genetic  method  in  order  to  determine  the  answers  to  
these  questions.  By  mutating  the  transformed  plants  with  the  miRNA  insert  through  
EMS  mutagenesis,  we  produced  a  great  variety  of  random  mutations.  By  observing  
the  relative  bioluminescence  of  these  mutated  plants,  we  can  then  track  the  
production  of  this  gene  and  determine  which  mutations  cause  an  increase  or  
decrease  in  its  expression.  As  the  study  continues,  we  will  continue  to  seek  the  
relationship  between  the  genes  and  biochemical  pathways  governing  this  process.    
Genes  are  crucial  in  everything  from  growth  and  development  to  responding  
to  environmental  stresses.  Since  irregular  gene  expression  is  essentially  the  cause  of  
disease,  determining  the  mechanisms  governing  its  regulation  is  paramount  to  

understanding  and  eventually  eliminating  disease.  Beyond  a  potential  application  to  
economically  important  crops,  miRNA  research  may  aid  in  developing  gene  therapy,  
regulating  gene  expression  caused  by  hereditary  disease  and  helping  to  eliminate  
disease  in  humans  as  well.