the gene expression pathway initiates at nuclear transcription generating a pre - mrna , which very often undergoes splicing , post - transcriptional regulation , and then translation into a protein . during the pre - mrna splicing process
, introns are removed and exons are joined in order to generate the mature mrna [ 13 ] .
the formation of this functional megacomplex is an orchestrated assembly of proteins and rna that requires identification of exon - intron boundaries .
exons are regularly alternatively spliced , meaning that they are either included or excluded from the final mature mrna transcript .
a recent comprehensive sequencing study observed that more than 90% of the genes undergo alternative splicing .
this vastly increases the transcriptome repertoire , and emphasizes both the significance of splicing and the requirement for its accurate execution .
in addition to protein coding genes , noncoding genes are transcribed . micrornas ( mirna ) , the most comprehensive noncoding group , are a class of   ~22 nt noncoding rnas that inhibit gene expression through binding to the 3 untranslated region ( utr ) of target mrna transcripts [ 6 , 7 ] .
there are hundreds of unique mirnas in a given species , each predicted to regulate a plethora of target genes [ 913 ] .
in fact , computational predictions indicate that mirnas may regulate 60% of all human protein coding genes .
therefore , it came as no surprise that mirnas were linked to many cellular processes such as differentiation , growth , and apoptosis , while mirna perturbations were associated with numerous diseases , including cancer [ 16 , 17 ] . in the past few years
, the pivotal role played by mirnas in gene regulation has been recognized [ 1820 ] .
mirnas are processed through a series of post - transcriptional biogenesis steps . the canonical maturation pathway , similar to protein - coding genes , initiates at transcription ( mostly by rna polymerase ii ) generating a primary ( pri- ) mirna .
the pri - mirna is characterized by a hairpin rna structure recognized by the nuclear rnase - iii enzyme drosha , and its cofactor dgcr8 .
the microprocessor cleaves the pri - mirna to generate a shorter hairpin of about 70 nt length  the pre - mirna .
this intermediate mirna is exported from the nucleus to the cytoplasm via   exportin-5 where the rnase iii endonuclease dicer generates the final mature mirna .
this short rna loses one of its strands ( the complementary mirna * strand ) while the other is loaded onto an argonaute - containing rna - induced silencing complex ( risc ) which mediates gene silencing .
once the mirna binds to its target gene , regulation takes place mainly through mrna degradation or translation inhibition [ 22 , 23 ] ( see figure 1 ) . for simplicity , the widely used term
mirnas can be located inter- or intragenically . when intergenic , their expression is coordinated with other mirnas as a cluster [ 25 , 26 ] .
when intragenic , namely , positioned within a protein - coding gene ( almost exclusively in introns ) , they are often expressed from the same strand as their host - gene [ 2730 ] and at correlated levels . given that both coding mrnas and mirnas are generated from the same transcriptional unit , and that they cooccur in close cellular proximity , it would be puzzling if these events exhibited total independence .
recent studies , from transcription , mirna - processing , and splicing oriented perspectives , have investigated these fascinating interactions and yielded interesting , yet somewhat controversial findings .
relationships between intronic mirnas and the processing events of their host mrna , namely , transcription and splicing have been addressed . here
we outline the major studies in the field .   expressed sequence tag ( est ) libraries of expressed mrnas
the ests represent a snapshot of cellular transcripts at a particular time point and thus display the given mrna plethora and its variety at a particular cellular state .
analysis of this data revealed several chimeric transcripts containing mirna and part of the adjacent mrna sequences . at an early stage of mirna research
notably , at a later stage , some of these est fragments were shown to be partially spliced , with either 5 or 3 ends matching putative drosha cleavage sites .
these results strengthened the possibility that mirnas and mrnas are processed from the same rna substrate .
in addition , the correlated expression pattern of host - gene transcripts and their mirnas   suggested that mirnas have coevolved to use the same promoter for transcription .
along with this work , by comparing mirna processing in a construct containing only the intronic sequence versus one that also includes the flanking exons , pawlicki and steitz   found that the levels of pri - mirna transcribed from introns are increased in the presence of flanking exons , due to prolonged retention at the site of transcription .
this supported the notion that flanking exons may facilitate mirna processing by increasing the time pri - mirnas spend tethered to the dna template [ 33 , 34 ] .
altogether , the data indicates that intronic mirna processing is enhanced by physical proximity to the site of transcription , and possibly also by splicing of the host gene .
several groups have isolated and identified various proteins associated with the human microprocessor complex [ 3537 ] . in these studies ,
numerous microprocessor - associated proteins were identified as splicing factors ( e.g. , hnrnph1 ; ) or involved in pre - mrna processing ( e.g. , dhx15 ; ) . taken together , based on mirna - mrna transcriptional ( est ) evidence ; shared promoters ; facilitated biogenesis when flanked by exons ; and overlapping proteins between the functional complexes , the data suggests that the microprocessor is potentially enhanced and present during transcription and most likely also during splicing .
if the same rna substrate is subjected to both host - gene and intronic mirna maturation , the intriguing question raised is how do all these processes  transcription , pre - mrna splicing , and mirna processing 
the complexity of the mirna - host - gene interaction model has increased recently when studies from the proudfoot laboratory demonstrated that pre - mirnas are generated through cotranscriptional cleavage by drosha .
suggested that efficient clearance of intronic sequences following microprocessor ( drosha ) cleavage may act to enhance the splicing efficiency .
these researchers showed that the microprocessor complex , as well as 5-3 and 3-5 rna exonucleases , are recruited to chromatin associated with intronic mirnas during transcription of the host primary transcript , and that drosha cleavage occurs before host intron splicing .
they found that mirna - harboring transcripts preferentially associated with chromatin fractions , from which they concluded that both pre - mirna cleavage and intronic splicing must occur on the same nascent transcripts .
the rapid exonucleolytic removal of intronic sequences may clear the proximal vicinity of rna processing for the purpose of efficiently completing the pre - mrna splicing task .
the enhancement of splicing by the microprocessor does not agree with other studies [ 30 , 40 ] ( discussed below ) and does not concur with experiments in yeast that showed enhanced processing for sirna flanked exons in splicing mutants .
dependencies are seen , for example , during the biogenesis process of small nucleolar rna ( snorna ) [ 4244 ] . in the process of snorna maturation , functional links between intronic snornp assembly ,
an antithesis to this dependency is the alternative mirna biogenesis pathway that bypasses the microprocessor via   generation of  mirtrons  [ 48 , 49 ] .
this mechanistically distinct class of intronic mirnas stem from very short introns where splicing substitutes the first step of mirna biogenesis . in this case , splicing activities replace the requirements for a microprocessor . however , not all roads lead to the observed dependency between microprocessing and splicing .
ying and lin   have designed an artificial intron containing a pre - mirna secondary structure .
they used this construct to show that the mature mirna was released only from the spliced intron .
this suggested that spliced introns are subsequently used by drosha and argued against any physical link between the microprocessor and the transcriptional unit or between the microprocessor and the spliceosome .
.   showed that exons of pre - mrna are tethered to the elongating rna polymerase ii either directly or indirectly without affecting processing , indicating that cotranscriptional cleavage of nascent intronic mirna transcripts does not affect splicing efficiency [ 40 , 51 ] . supporting the same view , kim and kim   addressed mirna biogenesis in light of the splicing mechanism .
they demonstrated that cleavage of an intronic mirna did not significantly affect the production of mature mrna and , conversely , the production of mature mirna was not significantly affected by splicing . in their experiments ,
knockdown of drosha , or mutations in the mirna hairpin , eliminated mirna generation without dramatically affecting mrna splicing .
this suggested that mirna biogenesis and splicing are coordinated but not functionally linked or interdependent . taking a closer look
, however , they also mention that drosha knockdown led to a modest increase in spliced mrna production and so did mutations in the mirna hairpin .
their work showed that the adjacent introns were spliced more rapidly than mirna - encoding introns , suggesting that binding of the microprocessor may eventually interfere with the splicing to some extent .
taken together , kim and kim 's data   imply mostly independent activities but can not exclude the possibility that the microprocessor interferes , to some level , with splicing . attempting to explain how both mirna and mrna are generated from the same dna locus , reconciling with the studies described here , we come up with two distinct models .
the second predicts generation of an mirna and mrna products arising from two independent rna transcripts ( see figure 2 ) . if the latter scenario was true , microprocessing and splicing would be independent of each other
. the outcome would be either no functional hindrance between the microprocessor and spliceosome activities or a competition for available pre - mrna substrates .
in the event that both rna products originate from the same precursor , it is conceivable that the two processes happen consecutively .
this would imply that execution of one process would be a prerequisite for the other to occur .
alternatively , mirna processing and mrna splicing may be coordinated so that the microprocessor and spliceosome interact with each other .
this interaction may be minimal , without affecting the amounts of mirna and mrna produced  as was suggested recently , or it may constitute a level of regulation .
we note that these events should always be looked at in the spatial - temporal context meaning that the microprocessor might act at an independent rate prior to the spliceosome assembly , and thus their direct interaction would be prevented . due to accumulating evidence , both in favor and against dependencies between splicing and microprocessing ,
the potential coordinated activity between the microprocessor and the spliceosome can be stratified into four possible relationships .
the microprocessor can inhibit or activate the spliceosome , and the spliceosome can inhibit or activate the microprocessor ( figure 3 ) .
, the microprocessor could activate the spliceosome by recruiting splicing factors to intronic mirnas , while at the same time the spliceosome could inhibit microprocessing .
we can not rule out , however , that these relationships occur in one large complex depending on the presence of particular rna processing proteins within the microprocessor .
many complex regulatory loops , both positive and negative , were seen in other cellular systems ( e.g. ,   see [ 52 , 53 ] ) .
the microprocessor , on the other hand , is composed of a handful of proteins [ 21 , 36 ] , minimally described as a two - protein complex [ 24 , 35 ] ( alternatively , see ) .
it is hard to visualize these two very differently - sized complexes aligned at the same position , competing for the same substrate .
thus , a coordinated processing and crosstalk seems necessary for these complexes  the microprocessor and the spliceosome  to be able to process the same transcript with intricate accuracy . during the mrna splicing process
, the rate at which transcription takes place may affect the transcripts ' pattern of splicing .
thus , kinetics of intron removal and exon ligation may play a role in selecting particular spliced isoforms .
given that some introns undergo mirna excision , unless the removal is extremely rapid , one can imagine a possible effect on splicing outcome .
thus , from an evolutionary perspective , an intronic mirna might evolve to participate in determining splicing kinetics .
consequently , an evolutionary driving force may direct mirna positioning within the intron to prevent disruption of relevant splice signals ( also see ) . in summary
how widespread is the mechanism and does it govern all intronic mirnas ? to date , not all microprocessor and spliceosome crosstalk scenarios ( as described in figure 3 ) have been identified . yet , given the complexity of cellular pathways , it is probably only a matter of time before their elucidation .
 micrornas ( mirnas ) are often hosted in introns of protein - coding genes . given that the same transcriptional unit can potentially give rise to both mirna and mrna transcripts 
 raises the intriguing question of the level of interaction between these processes . 
 recent studies from transcription , pre - mrna splicing , and mirna - processing perspectives have investigated these relationships and yielded interesting , yet somewhat controversial findings . here 
 we discuss major studies in the field . 