Cryptic unstable transcript
Encyclopedia
Cryptic Unstable Transcripts (CUTs) are a subset of non-coding RNA
s (ncRNAs) that are produced between intergenic and intragenic regions first observed in S. Cerevisiae yeast models and found in most eukaryote
s. CUTs belong to a class of RNA molecules transcribed by RNA Polymerase II
, but are quickly degraded and never expressed as proteins. Some basic characteristics of CUTs include a length of around 200-800 bp, a 5’ cap, poly-adenylated tail, and rapid degradation due to the combined activity of poly-adenylating polymerases and exosome complex
es. CUT transcription initiates from nucleosome
-depleted regions, often in an antisense orientation. To date, CUTs have a relatively uncharacterized function but have been found to participate in a number of putative gene regulation and silencing pathways. Thousands of loci leading to the generation of CUTs have been described in the yeast genome. Additionally, Stable Uncharacterized Transcripts, or SUTs, have also been detected in cells and bear many similarities to CUTs but are not degraded through the same pathways.
Regions of non-coding RNA were mapped in several early experiments examining S. Cerevisiae using a tiling array
approach, which indicated that a large amount of transcriptional activity could be attributed to the intergenic region of the genome. These transcripts are not readily observed in the mRNA population because they are rapidly targeted for degradation in both the nucleus and cytoplasm through a variety of mechanisms, but are able to be studied and characterized by examining yeast mutants with compromised enzyme capability which allow for CUT levels to be quantified.
In 2009, the Steinmetz and Jacquier laboratories performed a series of high-resolution transcriptome maps, further characterizing the widespread distribution and location non-coding transcripts within eukaryotes. CUTs were found to comprise around 13% of all mapped transcripts.
As CUTs cannot be observed at appreciable levels in wild-type S. Cerevisiae, a large component of their early study has focused on their degradation. To date, two main pathways have been identified: the recruitment of a degrading exosome via Nrd1-Nab3-Sen1 assisted by TRAMP, and termination due to the poly-adenylating capability of the Pap1p complex. In addition to these two main pathways, 5’ processing enzymes such as Xrn1 have also been shown to participate in CUT degradation. It was found that Δrrp6, a knock-out mutant for the exosome enzyme had heightened levels of transcripts mapped in transgenic regions. In fact, deletion of the RRP6 subunit has served as one of the earliest and most frequently used methods for generating high concentrations of CUTs in several experiments.
Stable Uncharacterized Transcripts or Stable Unannotated Transcripts (SUTs) share certain similar characteristics to CUTs- they can originate from the intergenic region, are non-coding transcripts, and undergo 5’ to 3’ cytoplasmic degradation. Like CUTs, SUT transcription start sites are also found at nucleosome free regions and are associated with promoters of protein coding genes. However, SUTs can be observed in both Δrrp6 mutants and wild type cells, indicating they are only partially degraded by the exosome and are able to escape the Nrd1-Nab3-Sen1 pathway. SUTs are primarily degraded instead by the combined activity of the decapping enzymes Dcp1, Dcp2 and the cytoplasmic exonuclease Xrn1.
CUT transcription has been shown to be suppressed in several histone-mediated regulatory mechanisms of gene expression. Within yeast models, it has been observed that the histone methyltransferase
Set2 is critical for maintaining proper acetylation
at Histone 3 Lysine 36 (H3K36). Loss of Set2 function results in over-acetylation, allowing for the expression of several short cryptic transcripts from genes STE11 and FLO8. In this case, the loss of Set2 allows for the expression of exon-derived CUTs as opposed to intergenic-derived transcripts, showing the role that histone regulation plays in controlling intragenic-derived CUTs. Similarly, in the absence of the transcription elongation factors Spt6 and Spt16, an incorrectly distributed nucleosome density allows for RNA Polymerase II to access cryptic polymerase sites, initiating erroneous transcription of CUTs. RNA Polymerase II has been observed to bind to interior initiation regions in the FLO8 gene in spt6 mutants, allowing for cryptic transcription to occur. Spt6 restores normal chromatin structure following transcription from RNA Polymerase II, and yeast cells with compromised Spt6 function have been found to have higher levels of gene expression but shorter transcript lengths, implicating the creation of CUTs. An antisense transcript located at the 3' end of PHO5 that is detectable in Δrrp6 mutants is responsible for increasing the speed of promoter remodeling, with mutants that lack the ability to transcribe the CUT having about half the rate of histone eviction as wild-type cells.
It has also been observed in S. Cerevisiae that Δrrp6 and Δtrf4 mutants have repressed transcription of the gene PHO84. Δrrp6 and Δtrf4 cells have stabilized levels of PHO84 antisense transcripts, which serve to recruit the Hda1/2/3 histone deacetylase
complex to the PHO84 gene, effectively silencing transcription and expression. It was found that in Δrrp6 cells Hda1 was associated with the promoter or coding regions of PHO84 up to five times more often when compared with wild-type counterparts, and that histone deacetylation activity occurred specifically at the region of PHO84 and Hda1 overlap on histone 3 lysine 18 (H3K18). Along with TY1 RTLs, PHO84 antisense transcripts can serve a potential regulatory function in S. Cerevisiae.
Promoter associated pervasive transcripts (PROMPTs) are found around 1-1.5 kb upstream of human transcription start sites in nongenic regions. Like CUTs, PROMPTs are a form of noncoding RNAs that become detectable in the absence of a degrading exosome enzyme. PROMPTs were first identified in siRNA-silenced hRrp40 human cells, where hRrp40 serves as a core subunit of the human exoribonucleoytic exosome. PROMPT-encoding regions have been found to produce sense and antisense transcripts, both of which are equally targeted by the exosome. In terms of function, ncRNAs with putative regulatory functions have been located to potential PROMPT regions. As a large portion of the human genome has been shown to be transcribed, the existence of PROMPTs helps explain a portion of the non-coding transcripts that are still generated.
Although an endogenous RNA interference
pathway does not exist within S. Cerevisiae, CUTs and SUTs may serve a comparable function. There has been an observed similarity between the suppression of the transposable element TY1 in yeast and small interfering RNA
activity within plants. In XRN1 mutants, TY1 transcripts decrease in number and TY1 antisense transcripts (TY1 RTLs) increase. TY1 RTLs reduce TY1 transposition activity in a trans manner and mitigates its expression, indicating a potential role for CUTs and SUTs in epigenetics
. Similarly, expression of the ncRNA SRG1 in S. Cerevisiae represses the transcriptional activity of the SER3 phosphoglycerate dehydrogenase gene. The rapidly degraded antisense transcripts of the gene PHO84 have also been shown to recruit the histone deacetylase Hda1 to the PHO84 gene as well, effectively suppressing PHO84 expression.
Non-coding RNA
A non-coding RNA is a functional RNA molecule that is not translated into a protein. Less-frequently used synonyms are non-protein-coding RNA , non-messenger RNA and functional RNA . The term small RNA is often used for short bacterial ncRNAs...
s (ncRNAs) that are produced between intergenic and intragenic regions first observed in S. Cerevisiae yeast models and found in most eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s. CUTs belong to a class of RNA molecules transcribed by RNA Polymerase II
RNA polymerase II
RNA polymerase II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase...
, but are quickly degraded and never expressed as proteins. Some basic characteristics of CUTs include a length of around 200-800 bp, a 5’ cap, poly-adenylated tail, and rapid degradation due to the combined activity of poly-adenylating polymerases and exosome complex
Exosome complex
The exosome complex is a multi-protein complex capable of degrading various types of RNA molecules...
es. CUT transcription initiates from nucleosome
Nucleosome
Nucleosomes are the basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound around a histone protein core. This structure is often compared to thread wrapped around a spool....
-depleted regions, often in an antisense orientation. To date, CUTs have a relatively uncharacterized function but have been found to participate in a number of putative gene regulation and silencing pathways. Thousands of loci leading to the generation of CUTs have been described in the yeast genome. Additionally, Stable Uncharacterized Transcripts, or SUTs, have also been detected in cells and bear many similarities to CUTs but are not degraded through the same pathways.
Discovery and Characterization of CUTS
Regions of non-coding RNA were mapped in several early experiments examining S. Cerevisiae using a tiling array
Tiling array
Tiling Arrays are a subtype of microarray chips. Like traditional microarrays, they function by hybridizing labeled DNA or RNA target molecules to probes fixed onto a solid surface. Tiling arrays differ from traditional microarrays in the nature of the probes...
approach, which indicated that a large amount of transcriptional activity could be attributed to the intergenic region of the genome. These transcripts are not readily observed in the mRNA population because they are rapidly targeted for degradation in both the nucleus and cytoplasm through a variety of mechanisms, but are able to be studied and characterized by examining yeast mutants with compromised enzyme capability which allow for CUT levels to be quantified.
In 2009, the Steinmetz and Jacquier laboratories performed a series of high-resolution transcriptome maps, further characterizing the widespread distribution and location non-coding transcripts within eukaryotes. CUTs were found to comprise around 13% of all mapped transcripts.
Degradation Pathways
As CUTs cannot be observed at appreciable levels in wild-type S. Cerevisiae, a large component of their early study has focused on their degradation. To date, two main pathways have been identified: the recruitment of a degrading exosome via Nrd1-Nab3-Sen1 assisted by TRAMP, and termination due to the poly-adenylating capability of the Pap1p complex. In addition to these two main pathways, 5’ processing enzymes such as Xrn1 have also been shown to participate in CUT degradation. It was found that Δrrp6, a knock-out mutant for the exosome enzyme had heightened levels of transcripts mapped in transgenic regions. In fact, deletion of the RRP6 subunit has served as one of the earliest and most frequently used methods for generating high concentrations of CUTs in several experiments.
The Nrd1-Nab3-Sen1 and TRAMP pathway
The observed increase of CUTs in Δrrp6 can be attributed to the activity of the Nrd1-Nab3-Sen1 pathway. Collectively, Nrd1, Nab3 and Sen1 bind to specific sequences of RNA and recruit the nuclear exosome which contains the degrading RRP6 subunit. Assisting in the Nrd1-Nab3-Sen1 pathway as a co-factor is the TRAMP complex, which is responsible for poly-adenylating transcripts and marking them for degradation. The TRAMP complex was discovered in Δrrp6 cells, when a certain population of poly-adenylated CUTs were attributed to the activity of a novel yeast polymerase, Trf4p, which was found to associate in a Trf4p-Air1p-Air2p complex. This collective complex (referred to as TRAMP) serves as an alternative Poly(A) polymerase to Pap1p within S. Cerevisiae.Role of Xrn1
Cytoplasmic decay of unstable transcripts can also be attributed to the activity of decapping enzymes and Xrn1. Transcripts that enter the cytoplasm can be targeted by the Dcp1-Dcp2 complex which removes the 5’ cap, allowing for the 5’ to 3’ exoribonuclease Xrn1 to degrade the transcript completely. The role of Dcp1-Dcp2 and Xrn1 in cytoplasmic decay has also been found to participate in the regulation of SUT levels.Relation with Bidirectional Promoters
Analysis of the transcription start sites of CUTs has found them to be within nucleosome free, non-overlapping transcript pairs. Nucleosome free regions of the genome have been frequently correlated with the promoter regions of open reading frames and mRNA transcripts, indicating that a portion of CUTs are located within bidirectional promoters. Additionally, the location of CUT 3’ ends are often found in close proximity to start features of ORFs in both sense and antisense configurations, with the end of sense CUT sequences laying within expressed proteins' 5’ promoter region. Sense CUTs have been found largely in promoters associated with glucose catabolism genes, while antisense CUTs have no association and are found dispersed in promoters across the entire genome.SUTs
Stable Uncharacterized Transcripts or Stable Unannotated Transcripts (SUTs) share certain similar characteristics to CUTs- they can originate from the intergenic region, are non-coding transcripts, and undergo 5’ to 3’ cytoplasmic degradation. Like CUTs, SUT transcription start sites are also found at nucleosome free regions and are associated with promoters of protein coding genes. However, SUTs can be observed in both Δrrp6 mutants and wild type cells, indicating they are only partially degraded by the exosome and are able to escape the Nrd1-Nab3-Sen1 pathway. SUTs are primarily degraded instead by the combined activity of the decapping enzymes Dcp1, Dcp2 and the cytoplasmic exonuclease Xrn1.
Interaction with Histones
CUT transcription has been shown to be suppressed in several histone-mediated regulatory mechanisms of gene expression. Within yeast models, it has been observed that the histone methyltransferase
Histone methyltransferase
Histone methyltransferases are enzymes, histone-lysine N-methyltransferase and histone-arginine N-methyltransferase, that catalyze the transfer of one to three methyl groups from the cofactor S-Adenosyl methionine to lysine and arginine residues of histone proteins...
Set2 is critical for maintaining proper acetylation
Acetylation
Acetylation describes a reaction that introduces an acetyl functional group into a chemical compound...
at Histone 3 Lysine 36 (H3K36). Loss of Set2 function results in over-acetylation, allowing for the expression of several short cryptic transcripts from genes STE11 and FLO8. In this case, the loss of Set2 allows for the expression of exon-derived CUTs as opposed to intergenic-derived transcripts, showing the role that histone regulation plays in controlling intragenic-derived CUTs. Similarly, in the absence of the transcription elongation factors Spt6 and Spt16, an incorrectly distributed nucleosome density allows for RNA Polymerase II to access cryptic polymerase sites, initiating erroneous transcription of CUTs. RNA Polymerase II has been observed to bind to interior initiation regions in the FLO8 gene in spt6 mutants, allowing for cryptic transcription to occur. Spt6 restores normal chromatin structure following transcription from RNA Polymerase II, and yeast cells with compromised Spt6 function have been found to have higher levels of gene expression but shorter transcript lengths, implicating the creation of CUTs. An antisense transcript located at the 3' end of PHO5 that is detectable in Δrrp6 mutants is responsible for increasing the speed of promoter remodeling, with mutants that lack the ability to transcribe the CUT having about half the rate of histone eviction as wild-type cells.
It has also been observed in S. Cerevisiae that Δrrp6 and Δtrf4 mutants have repressed transcription of the gene PHO84. Δrrp6 and Δtrf4 cells have stabilized levels of PHO84 antisense transcripts, which serve to recruit the Hda1/2/3 histone deacetylase
Histone deacetylase
Histone deacetylases are a class of enzymes that remove acetyl groups from an ε-N-acetyl lysine amino acid on a histone. This is important because DNA is wrapped around histones, and DNA expression is regulated by acetylation and de-acetylation. Its action is opposite to that of histone...
complex to the PHO84 gene, effectively silencing transcription and expression. It was found that in Δrrp6 cells Hda1 was associated with the promoter or coding regions of PHO84 up to five times more often when compared with wild-type counterparts, and that histone deacetylation activity occurred specifically at the region of PHO84 and Hda1 overlap on histone 3 lysine 18 (H3K18). Along with TY1 RTLs, PHO84 antisense transcripts can serve a potential regulatory function in S. Cerevisiae.
PROMPTs
Promoter associated pervasive transcripts (PROMPTs) are found around 1-1.5 kb upstream of human transcription start sites in nongenic regions. Like CUTs, PROMPTs are a form of noncoding RNAs that become detectable in the absence of a degrading exosome enzyme. PROMPTs were first identified in siRNA-silenced hRrp40 human cells, where hRrp40 serves as a core subunit of the human exoribonucleoytic exosome. PROMPT-encoding regions have been found to produce sense and antisense transcripts, both of which are equally targeted by the exosome. In terms of function, ncRNAs with putative regulatory functions have been located to potential PROMPT regions. As a large portion of the human genome has been shown to be transcribed, the existence of PROMPTs helps explain a portion of the non-coding transcripts that are still generated.
Function
Although an endogenous RNA interference
RNA interference
RNA interference is a process within living cells that moderates the activity of their genes. Historically, it was known by other names, including co-suppression, post transcriptional gene silencing , and quelling. Only after these apparently unrelated processes were fully understood did it become...
pathway does not exist within S. Cerevisiae, CUTs and SUTs may serve a comparable function. There has been an observed similarity between the suppression of the transposable element TY1 in yeast and small interfering RNA
Small interfering RNA
Small interfering RNA , sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a variety of roles in biology. The most notable role of siRNA is its involvement in the RNA interference pathway, where it...
activity within plants. In XRN1 mutants, TY1 transcripts decrease in number and TY1 antisense transcripts (TY1 RTLs) increase. TY1 RTLs reduce TY1 transposition activity in a trans manner and mitigates its expression, indicating a potential role for CUTs and SUTs in epigenetics
Epigenetics
In biology, and specifically genetics, epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence – hence the name epi- -genetics...
. Similarly, expression of the ncRNA SRG1 in S. Cerevisiae represses the transcriptional activity of the SER3 phosphoglycerate dehydrogenase gene. The rapidly degraded antisense transcripts of the gene PHO84 have also been shown to recruit the histone deacetylase Hda1 to the PHO84 gene as well, effectively suppressing PHO84 expression.