Structural basis of human U5 snRNP late biogenesis and recycling.

Pre-mRNA splicing by the spliceosome requires the biogenesis and recycling of its small nuclear ribonucleoprotein (snRNP) complexes, which are consumed in each round of splicing. The human U5 snRNP is the ~1 MDa 'heart' of the spliceosome and is recycled through an unknown mechanism involving major architectural rearrangements and the dedicated chaperones CD2BP2 and TSSC4. Late steps in U5 snRNP biogenesis similarly involve these chaperones. Here we report cryo-electron microscopy structures of four human U5 snRNP-CD2BP2-TSSC4 complexes, revealing how a series of molecular events primes the U5 snRNP to generate the ~2 MDa U4/U6.U5 tri-snRNP, the largest building block of the spliceosome.

mRNA recognition and packaging by the human transcription-export complex.

Newly made mRNAs are processed and packaged into mature ribonucleoprotein complexes (mRNPs) and are recognized by the essential transcription-export complex (TREX) for nuclear export1,2. However, the mechanisms of mRNP recognition and three-dimensional mRNP organization are poorly understood3. Here we report cryo-electron microscopy and tomography structures of reconstituted and endogenous human mRNPs bound to the 2-MDa TREX complex. We show that mRNPs are recognized through multivalent interactions between the TREX subunit ALYREF and mRNP-bound exon junction complexes. Exon junction complexes can multimerize through ALYREF, which suggests a mechanism for mRNP organization. Endogenous mRNPs form compact globules that are coated by multiple TREX complexes. These results reveal how TREX may simultaneously recognize, compact and protect mRNAs to promote their packaging for nuclear export. The organization of mRNP globules provides a framework to understand how mRNP architecture facilitates mRNA biogenesis and export.

Structure and properties of the giant reed (Arundo donax) lectin (ADL).

Arundo donax lectin (ADL) is a 170 amino acid protein that can be purified from the rhizomes of the giant reed or giant cane by exploiting its selective binding to chitin followed by elution with N-acetylglucosamine. The lectin is listed in the UniProt server, the largest protein sequence database, as an uncharacterized protein with chitin-binding domains (A0A0A9P802). This paper reports the purification, structure and ligand-binding properties of ADL. The lectin is a homodimer in which the two protomers are linked by two disulfide bridges. Each polypeptide chain presents four carbohydrate-binding modules that belong to carbohydrate-binding module family 18. A high degree of sequence similarity is observed among the modules present in each protomer. We have determined the X-ray structure of the apo-protein to a resolution of 1.70 Å. The carbohydrate-binding modules, that span a sequence of approximately 40 amino acids, present four internal disulfide bridges, a very short antiparallel central beta sheet and three short alpha helices, two on one side of the beta sheet and one on the other. The structures of the complexes of the lectin with N-acetylglucosamine, N-acetyllactosamine, N-acetylneuraminic acid and N-N'diacetylchitobiose reveal that ADL has two primary and two secondary carbohydrate-binding sites per dimer. They are located at the interface between the two protomers, and each binding site involves residues of both chains. The lectin presents structural similarity to the wheat germ agglutinin family, in particular, to isoform 3.

Structure of the human core transcription-export complex reveals a hub for multivalent interactions.

The export of mRNA from nucleus to cytoplasm requires the conserved and essential transcription and export (TREX) complex (THO-UAP56/DDX39B-ALYREF). TREX selectively binds mRNA maturation marks and licenses mRNA for nuclear export by loading the export factor NXF1-NXT1. How TREX integrates these marks and achieves high selectivity for mature mRNA is poorly understood. Here, we report the cryo-electron microscopy structure of the human THO-UAP56/DDX39B complex at 3.3 Å resolution. The seven-subunit THO-UAP56/DDX39B complex multimerizes into a 28-subunit tetrameric assembly, suggesting that selective recognition of mature mRNA is facilitated by the simultaneous sensing of multiple, spatially distant mRNA regions and maturation marks. Two UAP56/DDX39B RNA helicases are juxtaposed at each end of the tetramer, which would allow one bivalent ALYREF protein to bridge adjacent helicases and regulate the TREX-mRNA interaction. Our structural and biochemical results suggest a conserved model for TREX complex function that depends on multivalent interactions between proteins and mRNA.

The DNA of human and other eukaryotic cells is stored inside a compartment called the nucleus. DNA carries the genetic code and provides a blueprint for all of the cell's proteins. However, protein production occurs outside the nucleus, in the main body of the cell. To transmit genetic information from one compartment to the other, the DNA sequences are first transcribed into another molecule called messenger RNA, or mRNA for short. Once made, mRNA exits the nucleus and enters the cell's main body to encounter the machinery that translates its sequence into a protein. Before mRNA can exit the nucleus, it must first undergo a series of modifications, which result in the mRNA molecule being successively bound to specific proteins. Once mRNA has passed through these steps, it is recognized by the transcription-and-export complex, or TREX for short, which is comprised of several proteins. When TREX binds to mRNA, it adds on a final protein which allows the mRNA molecule to be transported out of the nucleus. However, it remained unclear how TREX selects the completed mRNA-protein complexes that are ready for export while at the same time recognizing the wide variety of mRNA molecules produced by cells. Now, Pühringer and Hohmann et al. have identified the first three-dimensional structure of the core of the human TREX complex using a technique called cryo-electron microscopy. This revealed that the seven proteins of the TREX core assemble into a large complex that has four copies of each protein. The structure suggests that TREX can bind to mRNA and its attached proteins in various ways. These different binding arrangements may help the complex select which mRNA molecules are fully modified and ready to be exported. The structure also sheds light on how mutations in this complex can lead to diseases such as Beaulieu­Boycott­Innes syndrome (BBIS). This work will help guide future research into the activity of TREX, including how its structure changes when it binds to mRNA and deposits the final transport protein. Identifying these structures will make it easier to design experiments that target specific aspects of TREX activity and provide new insights into how these complexes work.

The Cannabinoid Receptor Interacting Proteins 1 of zebrafish are not required for morphological development, viability or fertility.

The Cannabinoid Receptor Interacting Protein 1 (Cnrip1) was discovered as an interactor with the intracellular region of Cannabinoid Receptor 1 (CB1R, also known as Cnr1 or CB1). Functional assays in mouse show cannabinoid sensitivity changes and Cnrip1 has recently been suggested to control eye development in Xenopus laevis. Two Cnrip1 genes are described in zebrafish, cnrip1a and cnrip1b. In situ mRNA hybridisation revealed accumulation of mRNA encoding each gene primarily in brain and spinal cord, but also elsewhere. For example, cnrip1b is expressed in forming skeletal muscle. CRISPR/Cas9 genome editing generated predicted null mutations in cnrip1a and cnrip1b. Each mutation triggered nonsense-mediated decay of the respective mRNA transcript. No morphological or behavioural phenotype was observed in either mutant. Moreover, fish lacking both Cnrip1a and Cnrip1b both maternally and zygotically are viable and fertile and no phenotype has so far been detected despite strong evolutionary conservation over at least 400 Myr.