Background: The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A) tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the ciselements and produce the poly(A) tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis.

Animal models with genetic modifications under temporal and/or spatial control are invaluable to functional genomics and medical research. Here we report the generation of tissue-specific knockout rats via microinjection of zinc-finger nucleases (ZFNs) into fertilized eggs. We generated rats with loxP-flanked (floxed) alleles and a tyrosine hydroxylase promoter-driven cre allele and demonstrated Cre-dependent gene disruption in vivo. Pronuclear microinjection of ZFNs, shown by our data to be an efficient and rapid method for creating conditional knockout rats, should also be applicable in other species.

The protein Fip1 is an important subunit of the eukaryotic polyadenylation apparatus, since it provides a bridge of sorts between poly(A) polymerase, other subunits of the polyadenylation apparatus, and the substrate RNA. In this study, a previously unreported Arabidopsis The polyadenylation of messenger RNAs in the nucleus is an important step in the biogenesis of mRNAs in eukaryotes. This RNA processing reaction adds an essential cis element, the poly(A) tail, to the 3Ј-end of a processed pre-mRNA. This process is also coupled with many other steps in mRNA biogenesis (1). Thus, some polyadenylation factors are associated with transcription factors and recruit parts of the polyadenylation apparatus to the transcription initiation complex (2). Polyadenylation is linked to pre-mRNA splicing in a number of ways. For example, interactions between the polyadenylation and splicing machineries are important for the definition of 3Ј-terminal exons in animal cells (3, 4). Other interactions help to modulate different processing fates for pre-mRNAs, thus contributing to the scope of alternative splicing and polyadenylation in eukaryotes. The polyadenylation apparatus interacts with the C-terminal domain of the large subunit of RNA polymerase II (5-9) and with factors that play roles in transcription termination (10); these interactions suggest a central role for 3Ј-end processing in the termination of transcription by RNA polymerase II and subsequent recycling of polymerase II for new rounds of initiation.Polyadenylation is mediated by a multifactor complex in yeast and mammals. This complex recognizes the polyadenylation signal in the pre-mRNA, cleaves the pre-mRNA at a site that is defined by the cis elements, and adds a defined tract of poly(A) to the processed pre-mRNA. In mammals, the factors involved in this process have been classified according to chromatographic and biochemical behaviors, and termed cleavage and polyadenylation specificity factor (CPSF), 2 cleavage-stimulatory factor (CstF), and cleavage factors I and II (CFIm and CFIIm, respectively) (1). Each of these factors in turn consists of several distinct subunits. With the exception of CFIm (the two subunits of which are not obviously apparent in the yeast proteome), yeast possesses a similar array of polyadenylation factor subunits that form a somewhat different set of chromatographically distinct factors, namely cleavage and polyadenylation factor and cleavage factor I (1). Interestingly, the enzyme that adds poly(A) (poly(A) polymerase, or PAP) is part of the cleavage and polyadenylation factor in yeast nuclear extracts but fractionates largely as a separate protein in mammalian extracts. Whereas there are differences in the chromatographic behaviors of the complexes in mammals and yeast, most of the functions of the individual subunits seem to be similar. Besides the PAPs, this includes RNA binding by CPSF160, CPSF30, and CstF64 and their yeast counterparts (Yhh1p, Yth1p, and Rna15p, respectively) (11-17) and bridging between factors (CstF77 and it...

MAD7 is an engineered class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) system isolated from Eubacterium rectale. Analogous to Cas9, it is an RNA-guided nuclease with demonstrated gene editing activity in Escherichia coli and yeast cells. Here, we report that MAD7 is capable of generating indels and fluorescent gene tagging of endogenous genes in human HCT116 and U2OS cancer cell lines, respectively. In addition, MAD7 is highly proficient in generating indels, small DNA insertions (23 bases), and larger integrations ranging from 1 to 14 kb in size in mouse and rat embryos, resulting in live-born transgenic animals. Due to the different protospacer adjacent motif requirement, small-guide RNA, and highly efficient targeted gene disruption and insertions, MAD7 can expand the CRISPR toolbox for genome enginnering across different systems and model organisms.