The intron 1 of HPV 16 has a suboptimal branch point at a guanosine

Rosa-Rios, Marco Antonio De la; Martínez-Salazar, Martha; Martínez-García, Martha; González-Bonilla, César; Villegas‐Sepúlveda, Nicolás

doi:10.1016/j.virusres.2005.11.010

Cited by 9 publications

(10 citation statements)

References 49 publications

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“…Our result in Fig. 4C (lane 11) indicates that the nt 328G reported in other study [46] is not an authentic branch site. Interestingly, we observed a reduced E6*I splicing in the HEK293 cells transfected with the expression vector mt-4 containing adenosine mutations from the nt 383 to nt 385, where E6*I production level reduced to 62% (Fig.…”

Section: Resultsmentioning

confidence: 49%

“…In fact, the guanosine at nt 328 lacks a juxtaposed downstream PPT and has four additional AG dinucleotides upstream of the nt 409 3′ss. Thus, the proposed nt 328G as a branch site [46] does not fit into the splicing scanning model by recognizing the first AG dinucleotide 3′ of the branch site [35], [47]–[49]. Our result in Fig.…”

Section: Resultsmentioning

confidence: 64%

“…A G-to-C mutation at nt 328 position (Fig. 4B) was also constructed because the G nucleotide at the nt 328 was reported previously by another laboratory to be a putative branch site for splicing of the nt 409 3′ss [46]. After transfection of HEK293 cells, HPV16 E6E7 RNA splicing from each expression vector was profiled by RT-PCR using a primer pair of Pr106 and Pr855 as indicated in Fig.…”

Section: Resultsmentioning

confidence: 99%

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Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

et al. 2012

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HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5′ splice sites (5′ ss) and three 3′ splice sites (3′ ss) normally used in HPV16+ cervical cancer and its derived cell lines. The choice of two novel alternative 5′ ss (nt 221 5′ ss and nt 191 5′ ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5′ ss and nt 409 3′ ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3′ ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3′ ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of 91QYNK94 to 91PSFW94 displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.

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Section: Resultsmentioning

confidence: 49%

Section: Resultsmentioning

confidence: 64%

Section: Resultsmentioning

confidence: 99%

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Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

et al. 2012

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“…It is unclear, however, whether the single metal ion observed in the Dbr1 active site represents the native, active form of the enzyme or an incomplete active site that binds a second metal ion concomitant with the branched RNA substrate (see Materials and Methods). The low barrier hydrogen bond between Cys14 and the active site water nucleophile (Figure 2b) could facilitate the action of Cys14 as a catalytic base in a mononuclear mechanism, as the O2′ leaving group is thought to have a lower pK a than the O3′ leaving group (54). Additional studies are needed to fully elucidate Dbr1's catalytic mechanism.…”

Section: Discussionmentioning

confidence: 99%

Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1

Eric

Katolik

Clark

et al. 2014

Nucleic Acids Research

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The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2′,5′-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2′,5′-phosphodiester recognition and explain why the enzyme lacks activity toward 3′,5′-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents to treat retroviral infections and neurodegenerative disease.

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“…Thus, the coding sequences are recognized and spliced. Noteworthy, the alternatively spliced transcripts are heterogeneously expressed in cervical carcinoma cells [10,19,20].…”

Section: Introductionmentioning

confidence: 99%

Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16

Martínez-Salazar¹,

López–Urrutia²,

Aréchaga-Ocampo³

et al. 2014

Journal of Proteomics

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The intron 1 of HPV 16 has a suboptimal branch point at a guanosine

Cited by 9 publications

References 49 publications

Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1

Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16

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