Termination of pre-mRNA splicing requires that the ATPase and RNA unwindase Prp43p acts on the catalytic snRNA U6.

The termination of pre-mRNA splicing functions to discard suboptimal substrates, thereby enhancing fidelity, and to release excised introns in a manner coupled to spliceosome disassembly, thereby allowing recycling. The mechanism of termination, including the RNA target of the DEAH-box ATPase Prp43p, remains ambiguous. We discovered a critical role for nucleotides at the 3' end of the catalytic U6 small nuclear RNA in splicing termination. Although conserved sequence at the 3' end is not required, 2' hydroxyls are, paralleling requirements for Prp43p biochemical activities. Although the 3' end of U6 is not required for recruiting Prp43p to the spliceosome, the 3' end cross-links directly to Prp43p in an RNA-dependent manner. Our data indicate a mechanism of splicing termination in which Prp43p translocates along U6 from the 3' end to disassemble the spliceosome and thereby release suboptimal substrates or excised introns. This mechanism reveals that the spliceosome becomes primed for termination at the same stage it becomes activated for catalysis, implying a requirement for stringent control of spliceosome activity within the cell.

Mechanistic characterization of the 5'-triphosphate-dependent activation of PKR: lack of 5'-end nucleobase specificity, evidence for a distinct triphosphate binding site, and a critical role for the dsRBD.

The protein kinase PKR is activated by RNA to phosphorylate eIF-2α, inhibiting translation initiation. Long dsRNA activates PKR via interactions with the dsRNA-binding domain (dsRBD). Weakly structured RNA also activates PKR and does so in a 5'-triphosphate (ppp)-dependent fashion, however relatively little is known about this pathway. We used a mutant T7 RNA polymerase to incorporate all four triphosphate-containing nucleotides into the first position of a largely single-stranded RNA and found absence of selectivity, in that all four transcripts activate PKR. Recognition of 5'-triphosphate, but not the nucleobase at the 5'-most position, makes this RNA-mediated innate immune response sensitive to a broad array of viruses. PKR was neither activated in the presence of γ-GTP nor recognized NTPs other than ATP in activation competition and ITC binding assays. This indicates that the binding site for ATP is selective, which contrasts with the site for the 5' end of ppp-ssRNA. Activation experiments reveal that short dsRNAs compete with 5'-triphosphate RNAs and heparin for activation, and likewise gel-shift assays reveal that activating 5'-triphosphate RNAs and heparin compete with short dsRNAs for binding to PKR's dsRBD. The dsRBD thus plays a critical role in the activation of PKR by ppp-ssRNA and even heparin. At the same time, cross-linking experiments indicate that ppp-ssRNA interacts with PKR outside of the dsRBD as well. Overall, 5'-triphosphate-containing, weakly structured RNAs activate PKR via interactions with both the dsRBD and a distinct triphosphate binding site that lacks 5'-nucleobase specificity, allowing the innate immune response to provide broad-spectrum protection from pathogens.

Regulation of innate immunity through RNA structure and the protein kinase PKR.

Molecular recognition of RNA structure is key to innate immunity. The protein kinase PKR differentiates self from non-self by recognition of molecular patterns in RNA. Certain biological RNAs induce autophosphorylation of PKR, activating it to phosphorylate eukaryotic initiation factor 2α (eIF2α), which leads to inhibition of translation. Additional biological RNAs inhibit PKR, while still others have no effect. The aim of this article is to develop a cohesive framework for understanding and predicting PKR function in the context of diverse RNA structure. We present effects of recently characterized viral and cellular RNAs on regulation of PKR, as well as siRNAs. A central conclusion is that assembly of accessible long double-stranded RNA (dsRNA) elements within biological RNAs plays a key role in regulation of PKR kinase. Strategies for forming such elements include RNA dimerization, formation of symmetrical helical defects, A-form dsRNA mimicry, and coaxial stacking of helices.

Regulation of PKR by HCV IRES RNA: importance of domain II and NS5A.

Protein kinase R (PKR) is an essential component of the innate immune response. In the presence of double-stranded RNA (dsRNA), PKR is autophosphorylated, which enables it to phosphorylate its substrate, eukaryotic initiation factor 2alpha, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5'-untranslated region of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and noncanonical features of domain II cumulate to a total of approximately 33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV nonstructural protein 5A (NS5A) to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection.

A brilliant disguise for self RNA: 5'-end and internal modifications of primary transcripts suppress elements of innate immunity.

Interferon inducible protein kinase PKR is a component of innate immunity and mediates antiviral actions by recognizing pathogen associated molecular patterns (PAMPs). A well-known activator of PKR is long dsRNA, which can be produced during viral replication. Our recent results indicate that PKR can also be activated by short stem-loop RNA in a 5'-triphosphate-dependent fashion. A 5'-triphosphate is present primarily in foreign RNAs such as viral and bacterial transcripts, while a non-activating 5'-cap or 5'-monophosphate is present in most cellular RNAs. Additional studies indicate that internal RNA modifications and non-Watson-Crick motifs also repress PKR activation, and do so in an RNA structure-specific fashion. Interestingly, self-RNAs have more nucleoside modifications than non-self RNAs. Internal and 5'-end RNA modifications have repressive effects on other innate immune sensors as well, including TLR3, TLR7, TLR8, and RIG-I, suggesting that nucleoside modifications suppress innate immunity on a wide scale.

5'-triphosphate-dependent activation of PKR by RNAs with short stem-loops.

Molecular patterns in pathogenic RNAs can be recognized by the innate immune system, and a component of this response is the interferon-induced enzyme RNA-activated protein kinase (PKR). The major activators of PKR have been proposed to be long double-stranded RNAs. We report that RNAs with very limited secondary structures activate PKR in a 5'-triphosphate-dependent fashion in vitro and in vivo. Activation of PKR by 5'-triphosphate RNA is independent of RIG-I and is enhanced by treatment with type 1 interferon (IFN-alpha). Surveillance of molecular features at the 5' end of transcripts by PKR presents a means of allowing pathogenic RNA to be distinguished from self-RNA. The evidence presented here suggests that this form of RNA-based discrimination may be a critical step in mounting an early immune response.