in the 1950s , pulse - chase experiments using radioactive precursors showed that ~95% of the rna made in mammalian nuclei is degraded within minutes . at the time
, no credible reason could be proposed for this astonishing turnover , and only later did we discover it results from the destruction of non - coding rnas and intronic regions within coding transcripts .
while such synthesis and destruction appears utterly wasteful , it is nevertheless an integral part of metabolism . at the molecular level
for example , for every 100 transcripts initiated by a bacterial polymerase in vitro and in vivo , ~99 abort within ~10 nucleotides .
these aborting transcripts are not contained in current catalogs of transcripts made using rna - seq , as they are too short to be mapped .
eukaryotic polymerases then undergo a structural transition on  escape  from the promoter , but many stall and/or abort prematurely after 20500 nucleotides to give the promoter - proximal peak seen by rna - seq .
consequently , the production of each full - length sense transcript is accompanied by the synthesis of > 100 shorter products . in a human cell , the associated energy costs must be marginal , as typically nine - tenths of a message will now be spliced out and degraded .
unfortunately , there is little data on the half - lives of prematurely - terminated products , but it is likely they will be less than those of an intron ( typically ~5 min ) simply because they are so much shorter .
note that even the machines that we expect to perform precisely also initiate inefficiently ; most unique sequences in dividing human cells are present in equimolar amounts , but ~200-bp segments from origins of replication are in excess  and are presumably generated as dna polymerases abort .
( a ) strings of red spheres depict nucleotides ( nts ) incorporated into ~300 transcripts being copied from sense / anti - sense strands of a 15-kbp gene in a cell population .
most polymerases abort prematurely to yield short unstable transcripts of 210 nucleotides from both strands , a few pause / abort after generating unstable transcripts of 20500 nucleotides , and only one generates a stable full - length transcript .
the numbers of transcripts in each class are not known , and are included for illustrative purposes only ; however , there are probably so many short aborting ones of 210 nucleotides that the same general conclusion can be drawn irrespective of precise numbers .
( b ) after 20 ms ( when an elongating polymerase incorporates ~1 labeled nucleotide ; yellow ) , most incorporated label is in short transcripts that soon abort and are quickly degraded ; the long transcript contains one label , which goes undetected using most current methods .
( c ) after 5 min ( when a polymerase incorporates ~15  000 nucleotides ) , the one stable transcript is completely filled with label ; however , label incorporated early during the pulse into the many short aborting transcripts has turned over ( and so goes undetected ) .
imagine that we could see every nucleotide in every transcript copied from a typical human gene as it is incorporated during a 20-ms  pulse  ( when a polymerase adds ~1 nucleotide ) , and know what its fate might be ( fig . 
as the process is so inefficient , most labels at the end of the pulse will be in the many short transcripts that soon abort ( and are degraded rapidly , probably within seconds )
. only a tiny fraction will be in molecules that contribute to the stable transcriptome .
now imagine we repeat the experiment using a 5 min pulse ( long enough for a polymerase to transcribe the gene ; fig . 
all transcripts labeled early on will have been degraded , and the few contributing to the stable transcriptome will contain a much higher fraction of label ( because they are both stable and longer ) . as
traditional methods usually require pulses of > 5 min to allow detection of the full - length rna ( usually the one of interest ) , they completely miss the rapidly turning - over fraction .
in contrast to transcription , current reviews see a ribosome initiating efficiently , and then most being able to translate all the way to a termination codon without mishap  although some will stall to be rescued by pathways like those involved in  non - stop  and  no - go  decay .
most products made by ribosomes are also seen as stable , with the half - life of a typical human protein being ~20 h ( this average covers a wide range varying from minutes to tens of hours ) .
if production is efficient , and most products are stable , then the efficiency of translation must be very different from that of transcription !
genome sequences are annotated using complex assumptions , and it remains difficult to predict which segments encode open - reading frames ( orfs ) that are translated into protein , especially when those segments are short .
for example , it was initially assumed that orfs should be longer than 300 nucleotides , but ~10% of mouse orfs are shorter than this , and both ribosome profiling ( a method for monitoring ribosome binding along a message ) and proteomics show that many such short orfs ( sorfs ) are translated , whether they lie in genic or  non - coding  rnas .
about 50% of human mrnas also encode upstream open - reading frames ( uorfs ) of 9 nucleotides ( median length 48 nucleotides ) ; many of these uorfs are as evolutionarily conserved as orfs , and are translated into products that are present in 102,000 copies in the cell .
for example , translation of uorfs reduces expression of associated orfs , and mutations introducing a uorf into the 5 leader of the mrna encoding cdkn2a predisposes individuals to melanoma , in ldlr to familial hypercholesterolemia , and in cftr to cystic fibrosis .
some products of sorfs are also medically important , like gnrh1 in hormonal signaling , ccl / cxcl members in innate immunity , and defensins in pathogen protection .
only now are the dedicated pipelines needed for their detection being developed , with 74 new human peptides at the tip of the iceberg being found in a recent survey .
experiments using short pulses have uncovered an incredibly - high turnover of newly - made peptides .
these short pulses were being used in an attempt to solve an old chestnut  whether or not translation occurs in nuclei  and we now digress to discuss this . studies in the 1950s showed that isolated nuclei could incorporate radiolabeled amino acids into acid - insoluble protease - sensitive material , and this provoked a debate about whether this ( apparently nuclear ) synthesis was due to contamination by cytoplasmic ribosomes .
subsequently , it was shown that protein synthesis in bacteria is often spatially coupled to transcription , and it was expected it would be the same in eukaryotes . but
the discovery of introns seemed to provide a good reason why such coupling should not occur : if nuclear ribosomes translated introns  which possess many termination codons 
too many truncated peptides would be produced , and some would surely be toxic . clearly , restricting intron - containing rna to nuclei , and translation of intron - free mrna to the cytoplasm ,
this killed the idea that any translation might occur in nuclei . from time to time , the debate concerning nuclear translation has been re - opened . for example , translation is involved in proofreading mrna during nonsense - mediated decay ( nmd ) .
nmd involves the detection of inappropriately - placed termination codons in transcripts and degradation of the faulty rna .
as some nmd may occur in human nuclei , and as a translating ribosome is the only known mechanism able to detect stop codons , this points to some nuclear ribosomal activity .
( however , contrary findings indicate that nmd is predominantly cytoplasmic in yeast and mammals . )
thus , when hela cells are permeabilized and allowed to extend nascent peptides by ~15 residues in the presence of a translational precursor like biotin - lys - trna ( and triphosphates required for transcription ) , ~10% of the label in newly - made protein is nuclear ; this signal is reduced by incubation with transcriptional inhibitors , which suggests that some translation is biochemically coupled to transcription .
more recently , cells were pulsed for 5 min with puromycin ( a structural mimic of aminoacyl - trna ) , permeabilized , and fixed ; puromycin is incorporated by ribosomes into c - termini of nascent peptides , and it was then immuno - localized using an anti - puromycin antibody .
however , in both cases cells were permeabilized , and so critics can say cytoplasmic ribosomes were now able to enter nuclei to give rise to artifactual signals .
and although initiation factors , ribosomal proteins , and 80s ribosomes are all found in the nucleoplasm close to transcription sites , this might be expected : ribosomes are made in nucleoli and have to be exported through the nucleoplasm to get to the cytoplasm .
we can then summarize this digression as follows : since introns were discovered , the overwhelming consensus has been that all translation occurs in the cytoplasm .
baboo et al . revisited the question whether there was any nuclear translation using intact cells , and pulses too short to allow peptides made by cytoplasmic ribosomes to enter nuclei during the pulse .
azido - homoalanine ( aha ) is a met analog that possesses a reactive azide group , and aha - containing peptides can be localized after  clicking 
hela cells ( or primary diploid human cells ) were fed aha for periods as short as 5 s ( during which a ribosome in a living cell polymerizes ~25 residues , or ~one - sixteenth the length of a typical protein ) .
results obtained using a 2 min aha pulse are illustrated in figure  2 , and they are astonishing in two respects .
first , there is clear nuclear signal which appears brighter than the cytoplasmic one , although integration over the larger cytoplasmic area shows nuclei contain slightly less than half of all signals .
signals are reduced by pretreatment with translational inhibitors ( so ribosomes are involved ) , and increased by pretreatment with a proteasomal inhibitor ( so label is in protein ) .
second , signals in both compartments disappear during chases with half - lives of less than a minute , so that almost none remains after 5 min . figure  2 .
hela cells were starved of met for 15 min to deplete pools , pulsed for 2 min  2 mm aha , and chased ( 05 min ; 0.2 mm met without aha ) .
after fixation and  clicking  on alexa 555 , dna was counterstained with dapi , images collected using a wide - field microscope , and fluorescence intensities of alexa 555 (  sd ) seen during the chase in the cytoplasm and nucleus normalized relative to values at 0 min .
( b ) after a 2 min aha pulse , nuclei appear the brightest ; however , integration over the larger area of the cytoplasm indicates that this compartment contains slightly more signal .
( c ) if the 2 min pulse is followed by a 5 min chase , essentially no signal is seen .
( d ) alexa 555 fluorescence in both nucleus and cytoplasm declines rapidly during a chase . from baboo et al .
essentially similar results are obtained using two structurally - unrelated precursors  puromycin ( immunolocalized as above ) and heavy amino acids . in the latter case ,
amino acids were tagged with n ( or c ) , and distributions of incorporated n ( or c ) were mapped using a mass spectrometer attached to a microscope  an approach called nanosims ( high - resolution secondary - ion mass spectrometry ) . in a variant experiment
, hela cells were incubated for 2 min in both [ c]amino acids and [ n]amino acids ; then , ( cn ) ions derived from both nucleus and cytoplasm could be detected .
signals were again sensitive to ribosomal inhibitors , and decayed with half - lives of < 1 min .
this is consistent with the coupling by a ribosome of two different amino acids into one peptide bond , followed by the rapid destruction of that bond .
the answer is simple : we should only expect to detect it using a pulse that is roughly the length of the half - life , and such short pulses have not been used previously .
how can these results be reconciled with the known ~20 h half - life of the  mature  proteome ?
once we remember that this half - life was measured using pulses lasting days , the answer is again simple . just as only a tiny fraction of label incorporated during a short pulse is found in the one stable transcript in figure  1 , a 5 s or 2 min pulse should not be expected to label much of the mature proteome if a substantial amount of abortive translation and rapid turnover occurs .
but we should expect more labels to be incorporated into that mature proteome using longer pulses , and it is .
thus , if the experiment in figure  2c is repeated using 10 min and 1 h pulses , progressively more signals survive the 5 min chase .
finally , making a peptide is energetically costly , so how can the system support such waste ?
again the answer is simple : the cost must be marginal , as it is for transcription .
the extraordinary turnover is simply explained if the efficiency of translation is like that of transcription . just as the polymerization of one full - length transcript
is associated with an unproductive turnover of many shorter ones ( fig .  1 ) , we suggest that the polymerization of each full - length peptide is regulated by the synthesis of many shorter peptides that are rapidly degraded .
we envisage the scale of the peptide abortion has been underestimated , with perhaps only one in 20 initiated ribosomes translating all the way to a termination codon to generate a product that will contribute to the mature proteome .
we speculate that most recently - initiated ribosomes abort close to the beginning of uorfs , sorfs , and orfs because they fail to  escape  ( like an rna polymerase ) into a structure that allows stable elongation ( fig . 
3 ) ; then , the resulting short peptides ( combined with those made by translating to ends of many uorfs and sorfs ) are degraded rapidly . figure  3 . a model illustrating how dark - matter peptides ( green lines i - iii ) and the mature proteome ( iv ) might arise .
most initiated ribosomes terminate prematurely ( giving i and iii ) , and some translate to the end of the uorf ( giving ii ) ; the resulting peptides are rapidly degraded ( half - life < 1 min ) , to give rise to the astonishing turnover seen using short pulses .
a minority of ribosomes translates the whole orf ( giving iv ) ; such peptides are the ones detected conventionally using long pulses ( they are generally stable and contribute to the mature proteome ) . from baboo
this dark - matter world of aborting peptides has been hidden from our view for reasons much like those that had obscured dark - matter rna .
( 1 ) as illustrated in figure  1 , such a fraction can only be detected using a pulse time roughly the order of the half - life , and then so little label is incorporated that biochemical detection is challenging .
( 2 ) translation was traditionally monitored by incubation in radioactive amino acids , followed by protein precipitation with trichloroacetic acid and scintillation counting ; however di- and/or tri - peptides ( and probably longer ones too ) are not precipitated  and so were missed using this traditional approach .
short peptides are retained during the fixation used for microscopy , compared with the traditional acid extraction . )
( 3 ) short peptides are also missed during gel electrophoresis ; those with < 100 residues run together in most gels at the  front  , and those with < 10 remain unresolved even in dedicated gels . in both cases , they are difficult to detect because they bind proportionally less of any stain , and/or pass through membranes without binding during  blotting  .
( 4 ) if shorter than ~12 residues , they become indistinguishable from the background produced by proteasomes . ( 5 ) by analogy with a polymerase , a recently - initiated ribosome may bind less tightly to its mrna than one that has translated further  and so might be more likely to detach .
this could explain why ribosome profiling uncovers only a small peak of bound ribosomes near initiation codons ( as unstable ones detach ) , and why translation inhibitors increase the area of this peak  by  freezing  unstable ones on the message .
( 6 ) most peptides with < 6 peptides can not be mapped uniquely within the human proteome by mass spectrometry , and so are missed using current approaches .
the abortive synthesis and turnover probably play additional important roles including : ( 1 ) a nuclear ribosome might proofread a pre - mrna as it is being made to see if it has been spliced appropriately ( and so does not contain premature termination codons ) .
then , the faulty rna and truncated peptide produced as a by - product would be degraded 
( it was stated earlier that the discovery of introns killed the idea that translation might occur in nuclei because ribosomes must be prevented from translating intron - containing transcripts ; however , a nuclear ribosome would not  see  a termination codon in an intron if that ribosome was so placed it could only act on the transcript once splicing had occurred . )
( 2 ) if such nuclear proofreading of mrnas is error - prone , perhaps the system has a second go with a cytoplasmic ribosome at detecting a mis - spliced transcript once that transcript reaches the cytoplasm ( with consequential degradation of truncated peptides produced as by - products ) .
( 3 ) some mechanism must also exist to weed out the many non - coding transcripts like the anti - sense promoter transcripts ( which are immediately degraded ) from the few sense ones that go on to contribute to the stable transcriptome .
again , ribosomes acting co - transcriptionally as part of the nmd machinery could sense the difference between the two ( as non - coding transcripts probably contain many stop codons ) .
( 4 ) many more proteins than hitherto expected may misfold during synthesis , to be degraded immediately . ( 5 ) in the special case of antigen - presenting cells , some newly - made peptides may be processed rapidly , and the resulting fragments used in the defense against infection . in all these cases  whether it be nuclear ribosomes that specialize in proofreading newly - made transcripts , or cytoplasmic ones dedicated to mass - production 
the result is the same : inefficient translation , abortive production , and rapid degradation of resulting peptides .
the discovery of any new world always raises many questions . for example , how many different fractions of short peptides are there in a cell , how quickly does each one turn over , and what are the associated energy costs ?
critically , we also need to establish what roles the peptides in each fraction might play , and what additional effects the turnover might have .
thus , fluorescent tagging of proteins and super - resolution microscopy allow us to monitor interactions in living cells in ever - sharper detail .
moreover , we can use new bioinformatic pipelines to identify sorfs , ribosome profiling to see if they are translated , and 
however , we have seen that many challenges remain , especially in detecting and mapping short peptides . and like their counterparts in the rna world
 astrophysicists use the term  dark matter  to describe the majority of the matter and/or energy in the universe that is hidden from view , and biologists now apply it to the new families of rna they are uncovering . we review evidence for an analogous hidden world containing peptides . 
 the critical experiments involved pulse - labeling human cells with tagged amino acids for periods as short as five seconds . 
 results are extraordinary in two respects : both nucleus and cytoplasm become labeled , and most signals disappear with a half - life of less than one minute . just as the synthesis of each mature mrna 
 is regulated by the abortive production of hundreds of shorter transcripts that are quickly degraded , it seems that the synthesis of each full - length protein in the stable proteome is regulated by an apparently wasteful production and degradation of shorter peptides . 
 some of the nuclear synthesis is probably a byproduct of nuclear ribosomes proofreading newly - made rna for inappropriately - placed termination codons ( a process that triggers  nonsense - mediated decay  ) . we speculate that some  dark - matter  peptides will play other important roles in the cell . 
Inefficient Transcription and Rapid Turnover of Aborted Transcripts
Translation is Seen as Efficient and Proteins as Stable
The Proteome is Still Imprecisely Defined
A Digression on Nuclear Protein Synthesis
Astonishing Turnover of Newly-Made Peptides in Both Nucleus and Cytoplasm
A Molecular Explanation for the Peptide Turnover
Some Challenges