The Cap Snatching of Segmented Negative Sense RNA Viruses as a Tool to Map the Transcription Start Sites of Heterologous Co-infecting Viruses

ABSTRACT Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di- and trimeric forms. The mono- and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono- and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3′ and 5′ ends of the genomic RNA. In contrast, the mono- and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.

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Transcription shapes 3D chromatin organization by interacting with loop-extruding cohesin complexes

10.1101/2022.01.07.475367 ◽

2022 ◽

Author(s):

Edward J Banigan ◽

Wen Tang ◽

Aafke A van den Berg ◽

Roman R Stocsits ◽

Gordana Wutz ◽

...

Keyword(s):

Functional Organization ◽

Regulatory Elements ◽

General Shape ◽

Transcription Start ◽

Double Knockout ◽

Transcription Start Sites ◽

Contact Patterns ◽

New Type ◽

Active Transcription ◽

Distal Regulatory Elements

Cohesin organizes mammalian interphase chromosomes by reeling chromatin fibers into dynamic loops (Banigan and Mirny, 2020; Davidson et al., 2019; Kim et al., 2019; Yatskevich et al., 2019). "Loop extrusion" is obstructed when cohesin encounters a properly oriented CTCF protein (Busslinger et al., 2017; de Wit et al., 2015; Fudenberg et al., 2016; Nora et al., 2017; Sanborn et al., 2015; Wutz et al., 2017), and recent work indicates that other factors, such as the replicative helicase MCM (Dequeker et al., 2020), can also act as barriers to loop extrusion. It has been proposed that transcription relocalizes (Busslinger et al., 2017; Glynn et al., 2004; Lengronne et al., 2004) or interferes with cohesin (Heinz et al., 2018; Jeppsson et al., 2020; Valton et al., 2021; S. Zhang et al., 2021), and that active transcription start sites function as cohesin loading sites (Busslinger et al., 2017; Kagey et al., 2010; Zhu et al., 2021; Zuin et al., 2014), but how these effects, and transcription in general, shape chromatin is unknown. To determine whether transcription can modulate loop extrusion, we studied cells in which the primary extrusion barriers could be removed by CTCF depletion and cohesin's residence time and abundance on chromatin could be increased by Wapl knockout. We found evidence that transcription directly interacts with loop extrusion through a novel "moving barrier" mechanism, but not by loading cohesin at active promoters. Hi-C experiments showed intricate, cohesin-dependent genomic contact patterns near actively transcribed genes, and in CTCF-Wapl double knockout (DKO) cells (Busslinger et al., 2017), genomic contacts were enriched between sites of transcription-driven cohesin localization ("cohesin islands"). Similar patterns also emerged in polymer simulations in which transcribing RNA polymerases (RNAPs) acted as "moving barriers" by impeding, slowing, or pushing loop-extruding cohesins. The model predicts that cohesin does not load preferentially at promoters and instead accumulates at TSSs due to the barrier function of RNAPs. We tested this prediction by new ChIP-seq experiments, which revealed that the "cohesin loader" Nipbl (Ciosk et al., 2000) co-localizes with cohesin, but, unlike in previous reports (Busslinger et al., 2017; Kagey et al., 2010; Zhu et al., 2021; Zuin et al., 2014), Nipbl did not accumulate at active promoters. We propose that RNAP acts as a new type of barrier to loop extrusion that, unlike CTCF, is not stationary in its precise genomic position, but is itself dynamically translocating and relocalizes cohesin along DNA. In this way, loop extrusion could enable translocating RNAPs to maintain contacts with distal regulatory elements, allowing transcriptional activity to shape genomic functional organization.

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Reverse Genetics for Peste des Petits Ruminants Virus: Current Status and Lessons to Learn from Other Non-segmented Negative-Sense RNA Viruses

Virologica Sinica ◽

10.1007/s12250-018-0066-6 ◽

2018 ◽

Vol 33 (6) ◽

pp. 472-483 ◽

Cited By ~ 2

Author(s):

Alfred Niyokwishimira ◽

Yongxi Dou ◽

Bang Qian ◽

Prajapati Meera ◽

Zhidong Zhang

Keyword(s):

Reverse Genetics ◽

Rna Viruses ◽

Current Status ◽

Peste Des Petits Ruminants ◽

Negative Sense

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Expression of murine muscle-enriched A-type lamin-interacting protein (MLIP) is regulated by tissue-specific alternative transcription start sites

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.003758 ◽

2018 ◽

Vol 293 (51) ◽

pp. 19761-19770

Author(s):

Marie-Elodie Cattin ◽

Shelley A. Deeke ◽

Sarah A. Dick ◽

Zachary J. A. Verret-Borsos ◽

Gayashan Tennakoon ◽

...

Keyword(s):

Transcription Start ◽

Interacting Protein ◽

Tissue Specific ◽

Transcription Start Sites ◽

Alternative Transcription ◽

Specific Alternative

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Genomic organization of the 5′ end of human β-ENaC and preliminary characterization of its promoter

AJP Renal Physiology ◽

10.1152/ajprenal.00268.2001 ◽

2002 ◽

Vol 282 (5) ◽

pp. F898-F909 ◽

Cited By ~ 12

Author(s):

Christie P. Thomas ◽

Randy W. Loftus ◽

Kang Z. Liu ◽

Omar A. Itani

Keyword(s):

Genomic Organization ◽

Human Kidney ◽

Β Subunit ◽

Transcription Start ◽

Transcription Start Sites ◽

Nuclear Extracts ◽

Flanking Region ◽

Rapid Amplification ◽

Flanking Regions

The mRNA for the β-subunit of the epithelial Na+ channel (β-ENaC) is regulated developmentally and, in some tissues, in response to corticosteroids. To understand the mechanisms of transcriptional regulation of the human β-ENaC gene, we characterized the 5′ end of the gene and its 5′-flanking regions. Adaptor-ligated human kidney and lung cDNA were amplified by 5′ rapid amplification of cDNA ends, and transcription start sites of two 5′ variant transcripts were determined by nuclease protection or primer extension assays. Cosmid clones that contain the 5′ end of the gene were isolated, and analysis of these clones indicated that alternate first exons ∼1.5 kb apart and ∼ 45 kb upstream of a common second exon formed the basis of these transcripts. Genomic fragments that included the proximal 5′-flanking region of either transcript were able to direct expression of a reporter gene in lung epithelia and to bind Sp1 in nuclear extracts, confirming the presence of separate promoters that regulate β-ENaC expression.

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