NTR: [transcription pausing by RNA polymerase II]

[sylvainpoux]

Hi GO,

• While working on eralry steps of transcription, I realzed that there is not term for transcription pausing by RNA polymerase II, a step that is now recognized as an integral part of transcription. I will then reannotate the DSIF and NELF complexes, that are currently curated as inhibitors of transcription elongation, which is not exact, since they preceed transcription elongation. This step is also essential for RNA polymerase II transcription elongation surveillance (see ticket NTR: [RNA polymerase II transcription elongation surveillance] #29516)

Thanks

Sylvain

Term: transcription pausing by RNA polymerase II
Definition: A transcription halt following transcription initiation but prior to elongation, during which RNA polymerase II pauses approximately 20-60 nucleotides downstream of the transcriptional start site before proceeding into productive elongation.
Synonym: Promoter proximal pausing by RNA polymerase II
Child of GO:0006366 transcription by RNA polymerase II
PMID:35816930
PMID:33271312

We should also change the definition of transcription elongation
GO:0006368 transcription elongation by RNA polymerase II
FROM:
The extension of an RNA molecule after transcription initiation and promoter clearance at an RNA polymerase II promoter by the addition of ribonucleotides catalyzed by RNA polymerase II.
TO:
The extension of an RNA molecule after transcription pausing and promoter clearance at an RNA polymerase II promoter by the addition of ribonucleotides catalyzed by RNA polymerase II.

[ValWood]

perhaps the term label, if the term is added should be more specific, since transcriptional pausing by RNA polymerase II occurs in other contexts? (i.e termination)

[ValWood]

i.e use proposed exact synonym
promoter-proximal pausing by RNA polymerase II

Is the pause itself a process (I don't know)

NELF and DSIF prevent Pol II from entering productive elongation until phosphorylation by P-TEFb relieves the pause.

but these factors are involved in transcription elongation, so really is the way to capture this is that P-TEFb is involved in "positive/negative regulation of transcription elongation"? because until that signal ( to signify capping is complete, plus presumably an additional signal via the CTD code) elongation does not occur.

My favourite paper on this (I'm biased)
https://www.pombase.org/reference/PMID:19328067

In fact, the activatory/inhibitory mechanism is exactly the same for pausing at termination sites (controlled by P-TEFb (cdk9 in pombe) phosphorylating spt6
https://www.pombase.org/reference/PMID:29899453

I would wait for input from @pgaudet and @colinlog before adding. But it seems that the pausing is really controlled by the phosphorylation/dephosphorylation events controlled by the RNA polymerase II CTD-code mediated signalling.

[sylvainpoux]

Hi @ValWood
Yes, transcription pausing is now considered as a real process. This step acts as a quality control to check (1) if the elongation can take place or if (2) premature transcription termination of transcripts that are unfavorably configured should take place.

[ValWood]

it seems that the process that the pausing is related is a "checkpoint" between initiation and either elongation or surveillance. This would normally be modelled as a type of regulation.

[sylvainpoux]

Hi @ValWood
I see your point but the thing is that some protein complexes, such as DSIF act both during the transcription-pausing (they are a keymediator of this process) and also act as activator of transcription elongation. If we don't create a real term for transcription-pausing, it will be very confusing to have DSIF components that are both positive and negative regulator of transcription elongation. Moreover, we already have many GO terms for checkpoints.
That said, I agree that we should wait for @pgaudet and @colinlog
Sylvain

[ValWood]

I think you are correct, the process should exist in some way, but I wonder how it should be modelled? that's the tricky part. @pgaudet and @colinlog will have a better idea, I'm sure.

[krchristie]

Term: transcription pausing by RNA polymerase II
Definition: A transcription halt following transcription initiation but prior to elongation, during which RNA polymerase II pauses approximately 20-60 nucleotides downstream of the transcriptional start site before proceeding into productive elongation.

Hi @sylvainpoux - I did my PhD thesis on transcriptional pausing during the elongation phase and release from pausing by the elongation factor TFIIS. I took a quick look at one of the papers you cited (PMID:35816930) and it seems consistent with my memory.

Thus I think that the term name "transcription pausing by RNA polymerase II" that you are suggesting seems too specific for the definition you give which seems specific to "promoter escape and release into elongation". Transcriptional pausing may also occur further downstream than 60 nts from the transcriptional start site (TSS) during the productive elongation phase.

Another issue is that pauses can be intrinsic to the DNA structure or can be caused by DNA-binding proteins or DNA damage. Thus, depending on the kind of pause, there are proteins that promote pausing and others which stimulate elongation through pauses. Your definition currently seems to be a description that pausing can occur as part of promoter escape. However, it's not clear to me for what gene products this term is intended to be used for annotation, whether it's meant to be for gene products that promote the pause or for gene products that promote overcoming blocks to elongation.

[sylvainpoux]

@krchristie,

Thanks for the information. I'm fine if we propose another definition. Would you have any proposition?

I would need this term for
(1) proteins that mediate the pause (mainly DSIF and NELF complexes)
(2) proteins downstream of the pause: (2a) either elongation factors or (2b) Integrator complex, for transcripts that that are unfavorably configured for transcriptional elongation
(3) for proteins that release the pause, such as PPI1-PNUTs complex (negative regulation of transcription pausing by RNA polymerase II)

As mentioned before, most recent papers (from different groups) consider transcription pausing as a real step. My problem with transcription pausing described as regulation of transcription elongation is that it creates a real confusion for proteins that are involved both in transcription pausing and elongation (such as DSIF). It would also create confusions for the Integrator complex that terminates transcription, but which is upstream of transcription elongation.

This would be really difficult to create coherent models in GO-CAM.

Thanks

Sylvain

[krchristie]

@sylvainpoux - My first thought is that your items 1 & 3 should be represented by two different terms. I don't currently have a good sense of what item 2 is getting at in terms of a specific process or function.

I'll try to spend a little time thinking about this as I have been unsatisfied with the GO annotations for TFIIS since I started at SGD, but it has never been something I have been able to prioritize.

[sylvainpoux]

Thanks @krchristie !

I'm not sure that proteins that mediate and release the pause should be represented by two different terms in the sense that PPI1-PNUTs complex releases the pause by catalyzing dephosphorylation of proteins that promote the pause (DSIF complex).

Sylvain

[colinlog]

Hi Sylvain, Karen and Pascale, Indeed, much evidence suggests that every transcription event in eukaryotes involves a promoter proximal pause step that is imposed by DSIF and NELF. This occurs at the +1 nucleosome, which is decorated with H3K4me3 and involves the H2B ubiquitylation by the PAF complex. Pausing is a BP as multiple different MFs are involved. In my opinion, GO:0001109 promoter clearance during DNA-templated transcription] is the correct overarching (Prok-Archea+Euk) BP. From Wikipedia: DSIF is composed of hSPT4 <https://en.wikipedia.org/wiki/SUPT4H1> and hSPT5 <https://en.wikipedia.org/wiki/SUPT5H>.[2] <https://en.wikipedia.org/wiki/DSIF#cite_note-Wada-1998-2> hSPT5 has a direct role in mRNA capping which occurs while the elongation is paused. [4] <https://en.wikipedia.org/wiki/DSIF#cite_note-4> SPT5 is preserved in humans to bacteria.[5] <https://en.wikipedia.org/wiki/DSIF#cite_note-5> SPT4 and SPT5 in yeast are the homologs of hSPT4 and hSPT5.[2] <https://en.wikipedia.org/wiki/DSIF#cite_note-Wada-1998-2>[6] <https://en.wikipedia.org/wiki/DSIF#cite_note-6> In bacteria, the homologous complex only contains *NusG*, a Spt5 homolog.[7] <https://en.wikipedia.org/wiki/DSIF#cite_note-7> Archaea have both proteins. [8] <https://en.wikipedia.org/wiki/DSIF#cite_note-edge-8> The complex locks the RNA polymerase (RNAP) clamp into a closed state to prevent the elongation complex (EC) from dissociating. The Spt5 NGN domain <https://en.wikipedia.org/w/index.php?title=NGN_domain&action=edit&redlink=1> helps anneal the two strands of DNA upstream. The single KOW domain <https://en.wikipedia.org/w/index.php?title=KOW_domain&action=edit&redlink=1> in bacteria and archaea anchors a ribosome <https://en.wikipedia.org/wiki/Ribosome> to the RNAP.[8] <https://en.wikipedia.org/wiki/DSIF#cite_note-edge-8> .. One, new child term to describe the pausing, and it's relief, would be a new *BP: RNA polymerase II promoter-proximal pausing*. DSIF (and NELF in metazoa) are the main responsible proteins involved. This process is regulated by cyclin dependent kinase subfamily (CDK9 of pTEFb) which is (i) a transcription factor co-activator as well as (ii) a factor that must act at every gene promoter every time a new transcription round is initiated. Regulation is then about how fast/how often the pause is relieved. There is the question as to whether DSIF and NELF are also (anti)elongation factors.I think that TFIIS is actually a downstream target MF in the promoter escape and elongation process, involving the cleavage of the nascent transcript inside the paused RNA polymerase II complexes. In 2015, Koster and Timmers wrote: " Transcriptional pausing is a later evolutionary invention *(than initiation)*, and excellent reviews on this appeared recently (Adelman and Lis, 2012; Yamaguchi et al., 2013 <https://www.cell.com/cell/fulltext/S0092-8674(15)00489-4?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867415004894%3Fshowall%3Dtrue#>). " I think this strengthens the notion that it is a process rather than one (new) MF. A result of this action would be that the 'negative and positive regulation of transcription' annotations to DSIF and NELF subunits could be converted to a focussed step in transcription (promoter proximal pausing) and the regulation of that (sub)process would become accessible for annotation, which is key to the annotation of dbTFs and their co-TFs and the chromatin remodellers that are involved in transcription regulatory processes rather than the transcription biochemical processes (Initiation, promoter escape, elongation, termination) themselves? If you all agree, then Pascale and I could come up with a concrete proposal this Friday, unless Karen has already made a new proposal for the term requests by Sylvain by then? Best wishes, Colin

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On Mon, Jan 20, 2025 at 8:05 AM sylvainpoux ***@***.***> wrote: Thanks @krchristie <https://github.com/krchristie> ! I'm not sure that proteins that mediate and release the pause should be represented by two different terms in the sense that PPI1-PNUTs complex releases the pause by catalyzing dephosphorylation of proteins that promote the pause (DSIF complex). Sylvain — Reply to this email directly, view it on GitHub <#29529 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ALZVLKDYHI47HYVG6FWMDPL2LSN2TAVCNFSM6AAAAABU56DDE6VHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDMMBRGUZDQMBSG4> . You are receiving this because you were mentioned.Message ID: ***@***.***>