CR2 Flashcards
How did the authors prove that NMD evasion is not a result of translation repression? Describe their experimental approach and how the results were interpreted
They focused on the TRAM1 3’ UTR and the CRIPT 3’ UTR, using polysome profiling to test if the UTRs repressed polysome association. The polysome profile showed that the TRAM1 and CRIPT mRNAs were actively translated and associated with ribosomes. These results show that TRAM1 and CRIPT UTRs do not repress translation and polysome association. These UTRs are able to evade NMD while still being actively translated to ribosomes. This suggests that NMD evasion is not caused by translation repression.
Two potential mechanisms for evading NMD are suggested that invoke the cis elements characterized in this study. Describe in sufficient detail these two mechanisms
The first mechanism involves a subset of NMD-evading long 3′ UTRs, which contain NMD-inhibiting cis elements located within the first 200 nt. In TRAM1 3’ UTR, both the 5’ and the 3’ halves are capable of repressing NMD. This repression is dependent on being in downstream proximity to the termination codon. The first 82 nt appears to partially inhibit NDM when positioned in downstream proximity of the termination codon. There also seems to be a distance dependent NMD inhibiting cis element residing in the first 182 nt of the TRAM1 3’ UTR. This first 200 nt contains an element sufficient to inhibit NMD. The cis element might inhibit NMD in a number of possible ways.
- It could promote efficient translation termination
- it could promote a level of termination codon read through which has been previously associated with NMD evasion
- it could directly prevent the assembly or function of NMD factors on the long 3’ UTR mRNA.
TLDR: The cis elements located in the first 200 nt are A/U-rich, can inhibit NMD in a position-dependent manner and can promote NMD evasion when inserted into a heterologous NMD-sensitive mRNA.
The second mechanism appears to be different than the first one since their proximal 600 nt do not contain a cis element sufficient to promote NMD evasion. The 3’ UTRs might still have cis elements that can inhibit NMD, but just can’t do it in a heterologous context. The cis elements might reside more distally within the 3’ UTR. There is also some evidence that NMD inhibition might be caused by the structural arrangement of the long 3’ UTR that brings PABP in spatial proximity to the termination codon.
. Two of the mRNA transcripts in Fig. 1 show increases in RNA expression following depletion of UPF1. What are these two transcripts and why does their expression increase while the other transcripts stay stable or decline
The two transcripts are GABARAPL1 mRNA and SMG5 mRNA. These mRNAs have been formerly considered as NMD targets. UPF1 Depletion resulted in increases in steady state levels of SMG5 and GABARAPL1. UPF1 is a protein important in NMD, if you knock out the UPF1 with a targeted siRNA then there should be no NMD. In figure one they’ve knocked out the UPF1 from all the mRNAs. They’re looking at the recovery of transcription after they’ve knocked out UPF1. If NMD was active there should be decay and when you knock out UPF1 it should recover. The first 2 recovered which are SMG5 and GAGARAPL1. This means these two were in fact targeted by NMD. These other 6 of the mRNAs shown in the figure didn’t show any recovery at all. This means they were never targeted by NMD and therefore never started to decay at all so there was nothing to recover from.
The observation is consistent with the notion that many endogenous mRNAs with long 3’UTRs are not targeted by NMD pathway despite artificial long 3’UTRs activating NMD. If the 3’ UTRs are inserted into βWT reporter mRNA it resulted in destabilization and increased half lives upon UPF1 Depletion, further showing that GABARAPL1 mRNA and SMG5 mRNA are targeted by NMD. The other mRNAs seen in figure 1 are resistant to NMD and didn’t trigger NMD when they were inserted into the βWT reporter mRNA, as seen by the lack of stabilization upon UPF1 Depleting. Some of the 3’UTRs dud destabilize the βWT mRNA, but these mRNAs did so independent of NMD. These 3’ UTRs likely have features that prevent the activation of NMD.
The authors chose to use UPF1 depletion as a measure of NMD activity. Explain why this is a good assay for NMD activity. Name two additional proteins that could have been chosen for depletion that might have served the same purpose as UPF1 and explain the basis for your answer
UPF2, upf3, specific proteins more specific to NMD
premature termination codons (PTCs)
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The NMD substrates are detected by proteins such as UPF1 after translation gets stopped at a premature stop codon.1 The UPF1 protein recognizes the PTC-containing mRNAs because of the exon-junction complex remaining on transcripts with premature stop codons.2 UPF1 assembles with NMD substrates forming a complex that targets PTC-containing mRNA for degradation.1 Therefore, this is a good measure of NMD activity because as UPF1 is depleted, there should also be less NMD. Phosphorylation and dephosphorylation of UPF1 by SMG proteins is essential to NMD.3 Phosphorylation of UPF1 by SMG proteins is needed to identify the exon-junction complexes on PTC-containing mRNA.
Based on their results the authors describe 3 classes of human 3’UTRs. Describe the basis for this classification and give example mRNAs for each class
The three classes were found by looking to see what mRNAs did or did not render their mRNA insensitive to NMD. This was done by comparing the mRNA to ßWT-GFP when they were inserted downstream from the termination codon in ßWT-GFP mRNA. If the mRNA was stable and insensitive to UPF1 depletion then the NMD was inhibited. If the mRNA was unstable it did not render the mRNA insensitive to NMD.
- The first of the three classes renders their mRNA sensitive to NMD. This composes of SMG5, MFN2, AP2A2 and GABARAPL1 3′ UTRs. The first 200 nt of these do not render ßWT-GFP mRNA insensitive to NMD.
- The next class is resistant to NMD from the presence of sequence elements. These inhibit NMD in a manner dependent on their proximity to the termination codon. Their first 200 nt all resulted in mRNA stabilization as well as depletion if they were inserted immediately downstream from the termination codon. Some examples include TMED2, VAMP3, TRAM1 and CRIPT.
- Last of all is the third class which is also NMD resistant. This class however has unknown mechanisms involved in the NMD resistance. It’s first 200, 400 and 600 nt were not sufficient to inhibit NMD of the ßWT-GFP reporter mRNA. mRNA examples of this are PSMD5 and PRKAB1.
This article focuses on human mRNA transcripts but other studies have described how some viruses can evade NMD. What cis-acting element in the Rous Sarcoma virus permits it to evade NMD
The Rous Sarcoma virus produces unspliced retroviral transcripts that contain many NMD-activating features. These transcripts are protected from NMD by the “presence of a 400 nt cis-acting element located downstream from the gag ORF”. The RNA stability element (RSE) is the 400 nt element which is downstream of the gag termination codon in the unsliced Rous Sarcoma virus. In chicken cells, the RSE is shown to protects the viral RNA from Up-frameshift protein 1 (UPF1) dependent decay. Due to truncations, a minimal RSE element of 155 nt in the RSE is found.
While trans acting factors are presumed to bind to the A/U rich regions identified by the authors, what alternative structural scheme could describe the role for the A/U region in NMD evasion
The paper states that there is a possibility that a trans factor with affinity for A/U rich regions may mediate the NMD inhibitory effects of the cis elements. One method for this activity is could be PABP, which has been known to show high affinity for A/U-rich regions and is a known antagonist of NMD. Another option is that RNA binding proteins that are involved in either translational termination of NMD factor function, or that recruit other factors such as PABP, could be responsible. Another method is that the A/U-rich cis elements base-pair with the poly(A) tail, therefore bringing the poly(A) tail, and hence PABP, near the termination event. To add, a recent study showed evidence that UPF1 associates most stable with G/C rich regions. Therefore, the A/U rich composition of the cis elements could stop stable UPF1 binding in downstream proximity of the termination event.
