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Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7.

ABSTRACT: The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3'-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3' hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5'-end with the foot of the 3' hairpin and has thus functional implications.

SUBMITTER: Uchikawa E

PROVIDER: S-EPMC4381077 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7.

Uchikawa Emiko E Natchiar Kundhavai S KS Han Xiao X Proux Florence F Roblin Pierre P Zhang Elodie E Durand Alexandre A Klaholz Bruno P BP Dock-Bregeon Anne-Catherine AC

Nucleic acids research 20150309 6

The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3'-end of 7SK. The structure sh ...[more]

PMID: 25753663

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Project description:Species of the Legionella genus encode at least 18,000 effector proteins that are translocated through the Dot/Icm type IVB translocation system into macrophages and protist hosts to enable intracellular growth. Eight effectors, including ankyrin H (AnkH), are common to all Legionella species. The AnkH effector is also present in Coxiella and Rickettsiella To date, no pathogenic effectors have ever been described that directly interfere with host cell transcription. We determined that the host nuclear protein La-related protein 7 (LARP7), which is a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, interacts with AnkH in the host cell nucleus. The AnkH-LARP7 interaction partially impedes interactions of the 7SK snRNP components with LARP7, interfering with transcriptional elongation by polymerase (Pol) II. Consistent with that, our data show AnkH-dependent global reprogramming of transcription of macrophages infected by Legionella pneumophila The crystal structure of AnkH shows that it contains four N-terminal ankyrin repeats, followed by a cysteine protease-like domain and an α-helical C-terminal domain. A substitution within the β-hairpin loop of the third ankyrin repeat results in diminishment of LARP7-AnkH interactions and phenocopies the ankH null mutant defect in intracellular growth. LARP7 knockdown partially suppresses intracellular proliferation of wild-type (WT) bacteria and increases the severity of the defect of the ΔankH mutant, indicating a role for LARP7 in permissiveness of host cells to intracellular bacterial infection. We conclude that the AnkH-LARP7 interaction impedes interaction of LARP7 with 7SK snRNP, which would block transcriptional elongation by Pol II, leading to host global transcriptional reprogramming and permissiveness to L. pneumophilaIMPORTANCE For intracellular pathogens to thrive in host cells, an environment that supports survival and replication needs to be established. L. pneumophila accomplishes this through the activity of the ∼330 effector proteins that are injected into host cells during infection. Effector functions range from hijacking host trafficking pathways to altering host cell machinery, resulting in altered cell biology and innate immunity. One such pathway is the host protein synthesis pathway. Five L. pneumophila effectors have been identified that alter host cell translation, and 2 effectors have been identified that indirectly affect host cell transcription. No pathogenic effectors have been described that directly interfere with host cell transcription. Here we show a direct interaction of the AnkH effector with a host cell transcription complex involved in transcriptional elongation. We identify a novel process by which AnkH interferes with host transcriptional elongation through interference with formation of a functional complex and show that this interference is required for pathogen proliferation.

Dataset Information

Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7.

Publications

Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7.

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