Cryo-EM structure of the CBC-ALYREF complex.

In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5' end with a 7-methylguanosine (m 7 G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism including transcription, splicing, polyadenylation and export. It promotes mRNA export through direct interaction with ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5' end of mRNA. However, the molecular mechanism for CBC mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with a mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contacts with both the NCBP1 and NCBP2 subunits of the CBC. Comparison of CBC-ALYREF to other CBC and ALYREF containing cellular complexes provides insights into the coordinated events during mRNA transcription, splicing, and export.

Structural basis for high-order complex of SARNP and DDX39B to facilitate mRNP assembly.

mRNA in eukaryotic cells is packaged into highly compacted ribonucleoprotein particles (mRNPs) in the nucleus and exported to the cytoplasm for translation. mRNP packaging and export require the evolutionarily conserved transcription-export (TREX) complex. TREX facilitates loading of various RNA-binding proteins on mRNA through the action of its DDX39B subunit. SARNP (Tho1 [transcriptional defect of Hpr1 by overexpression 1] in yeast) is shown to interact with DDX39B and affect mRNA export. The molecular mechanism of how SARNP recognizes DDX39B and functions in mRNP assembly is unclear. Here, we determine the crystal structure of a Tho1/DDX39B/RNA complex, revealing a multivalent interaction mediated by tandem DDX39B interacting motifs in SARNP/Tho1. The high-order complex of SARNP and DDX39B is evolutionarily conserved, and human SARNP can engage with five DDX39B molecules. RNA sequencing (RNA-seq) from SARNP knockdown cells shows the most affected RNAs in export are GC rich. Our work suggests the role of the high-order SARNP/DDX39B/RNA complex in mRNP assembly and export.

Cryo-EM structure of the yeast TREX complex and coordination with the SR-like protein Gbp2.

The evolutionarily conserved TRanscript-EXport (TREX) complex plays central roles during mRNP (messenger ribonucleoprotein) maturation and export from the nucleus to the cytoplasm. In yeast, TREX is composed of the THO sub-complex (Tho2, Hpr1, Tex1, Mft1, and Thp2), the DEAD box ATPase Sub2, and Yra1. Here we present a 3.7 Šcryo-EM structure of the yeast THO•Sub2 complex. The structure reveals the intimate assembly of THO revolving around its largest subunit Tho2. THO stabilizes a semi-open conformation of the Sub2 ATPase via interactions with Tho2. We show that THO interacts with the serine-arginine (SR)-like protein Gbp2 through both the RS domain and RRM domains of Gbp2. Cross-linking mass spectrometry analysis supports the extensive interactions between THO and Gbp2, further revealing that RRM domains of Gbp2 are in close proximity to the C-terminal domain of Tho2. We propose that THO serves as a landing pad to configure Gbp2 to facilitate its loading onto mRNP.

Structure and activation mechanism of the yeast RNA Pol II CTD kinase CTDK-1 complex.

The C-terminal domain (CTD) kinase I (CTDK-1) complex is the primary RNA Polymerase II (Pol II) CTD Ser2 kinase in budding yeast. CTDK-1 consists of a cyclin-dependent kinase (CDK) Ctk1, a cyclin Ctk2, and a unique subunit Ctk3 required for CTDK-1 activity. Here, we present a crystal structure of CTDK-1 at 1.85-Å resolution. The structure reveals that, compared to the canonical two-component CDK-cyclin system, the third component Ctk3 of CTDK-1 plays a critical role in Ctk1 activation by stabilizing a key element of CDK regulation, the T-loop, in an active conformation. In addition, Ctk3 contributes to the assembly of CTDK-1 through extensive interactions with both Ctk1 and Ctk2. We also demonstrate that CTDK-1 physically and genetically interacts with the serine/arginine-like protein Gbp2. Together, the data in our work reveal a regulatory mechanism of CDK complexes.

Cellular mRNA export factor UAP56 recognizes nucleic acid binding site of influenza virus NP protein.

Influenza A virus nucleoprotein (NP) is a structural component that encapsulates the viral genome into the form of ribonucleoprotein complexes (vRNPs). Efficient assembly of vRNPs is critical for the virus life cycle. The assembly route from RNA-free NP to the NP-RNA polymer in vRNPs has been suggested to require a cellular factor UAP56, but the mechanism is poorly understood. Here, we characterized the interaction between NP and UAP56 using recombinant proteins and showed that UAP56 features two NP binding sites. In addition to the UAP56 core comprised of two RecA domains, we identified the N-terminal extension (NTE) of UAP56 as a previously unknown NP binding site. In particular, UAP56-NTE recognizes the nucleic acid binding region of NP. This corroborates our observation that binding of UAP56-NTE and RNA to NP is mutually exclusive. Collectively, our results reveal the molecular basis for how UAP56 acts on RNA-free NP, and provide new insights into NP-mediated influenza genome packaging.