RNA interference (RNAi) has become a research tool to control gene expression in various organisms and holds potential as a new therapeutic strategy. The mechanism of small interfering RNA (siRNA)-mediated RNAi involves target mRNA cleavage and destruction in the cytoplasm. We investigated siRNA-mediated induction of RNAi in the nucleus of human cells. Notably, we observed highly efficient knockdown of small nuclear RNA 7SK by siRNA. siRNA- and microRNA-programmed RNA-induced silencing complexes (RISCs) were present in both cytoplasmic and nuclear compartments and specifically cleaved their perfectly matched target RNA with markedly high efficiencies. Our results provide the first evidence that human RISCs programmed with siRNA are present in the nucleus and can knock down target RNA levels. These studies reveal new roles for the RNAi machinery in modulating post-transcriptional gene expression in the nucleus.

At least half of all human pre-mRNAs are subject to alternative 3 processing that may modulate both the coding capacity of the message and the array of post-transcriptional regulatory elements embedded within the 3 UTR. Vertebrate poly(A) site selection appears to rely primarily on the binding of CPSF to an A(A/U)UAAA hexamer upstream of the cleavage site and CstF to a downstream GU-rich element. At least one-quarter of all human poly(A) sites, however, lack the A(A/U)UAAA motif. We report that sequence-specific RNA binding of the human 3 processing factor CFI m can function as a primary determinant of poly ( The process of mRNA 3Ј end formation is not simply a perfunctory step in eukaryotic gene expression. At least one-half of all human genes are subject to alternative 3Ј processing (Iseli et al. 2002), the consequences of which may impact the protein coding capacity of the message, as well as its localization, translation efficiency, and stability (Edwalds-Gilbert et al. 1997). Moreover, poly(A) site selection may be modulated in a developmental and tissue-specific manner. In addition, pre-mRNA 3Ј processing contributes directly to transcription termination (Zorio and Bentley 2004), pre-mRNA splicing , and mRNA export (Hammell et al. 2002;Lei and Silver 2002). While the processing of constitutive poly(A) sites has been examined in considerable detail, the fundamental mechanisms responsible for the regulation of alternative poly(A) site selection have yet to be fully elucidated (Barabino and Keller 1999).The processing of the majority of human poly(A) sites involves the recognition of an AAUAAA or AUUAAA hexamer by CPSF, coupled with the binding of CstF to a GU-rich downstream element (DSE) (Zhao et al. 1999). The binding of CPSF and CstF appears to be sufficient, at least in vitro, to direct the assembly of a 3Ј processing complex composed of at least 14 different proteins. In vivo, however, the hexamer and DSE alone are unlikely to suffice for poly(A) site definition. The recognition of an authentic poly(A) site within a nascent RNA in vivo appears to rely on the "biosynthetic context" provided by the transcription elongation complex (Proudfoot 2004). At least nine 3Ј processing proteins are recruited to the transcription complex, at least in part through interactions with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) (Calvo and Manley 2003). The colocalization of 3Ј processing factors, along with capping enzymes and spliceosome components, to the transcription elongation complex, allows for the cooperative interaction of these processing machineries within an "mRNA factory" (Zorio and Bentley 2004).Cotranscriptional recognition of a poly(A) site provides an elegant mechanism for the identification of a processing site demarcated by a limited set of sequence motifs. Yet the mechanisms that regulate the selection of alternative poly(A) sites within a pre-mRNA, or allow for the recognition of poly(A) sites that lack the canonical A(A/U)UAAA motif, are poorly understood. Seque...

The complex formed by the hairpin ribozyme and its substrate consists of two independently folding domains which interact to form a catalytic structure. Fluorescence resonance energy transfer methods permit us to study reversible transitions of the complex between open and closed forms. Results indicate that docking of the domains is required for both the cleavage and ligation reactions. Docking is rate-limiting for ligation (2 min-1) but not for cleavage, where docking (0.5 min-1) precedes a rate-limiting conformational transition or slow-reaction chemistry. Strikingly, most modifications to the RNA (such as a G+1A mutation in the substrate) or reaction conditions (such as omission of divalent metal ion cofactors) which inhibit catalysis do so by preventing docking. This demonstrates directly that mutations and modifications which inhibit a step following substrate binding are not necessarily involved in catalysis. An improved kinetic description of the catalytic cycle is derived, including specific structural transitions.

Human cleavage factor I(m) (CFI(m)) is a heterodimeric RNA binding protein complex that functions at an early step in the assembly of the pre-mRNA 3' processing complex. In this report we show that CFI(m) can stimulate both cleavage and poly(A) addition, and can act to suppress poly(A) site cleavage in a sequence-dependent manner. Elevated levels of CFI(m) suppressed cleavage at the primary poly(A) site of the pre-mRNA encoding the 68 kDa subunit of CFI(m). CFI(m)-mediated suppression of poly(A) site cleavage was dependent upon the presence of three copies of an RNA element initially identified by CFI(m)-SELEX. These data provide evidence for a mechanism for the regulation of poly(A) site selection by a basal pre-mRNA 3' processing factor.

We present a series of 14 binary vectors suitable for Agrobacterium-mediated transformation of dicotyledonous plants and adaptable for biolistic transformation of monocotyledonous plants. The vector size has been minimized by eliminating all non-essential elements from the vector backbone and T-DNA regions while maintaining the ability to replicate independently. The smallest of the vector series is 6.3 kb and possesses an extensive multiple cloning site with 21 unique restriction endonuclease sites that are compatible with common cloning, protein expression, yeast two-hybrid and other binary vectors. The T-DNA region was engineered using a synthetic designer oligonucleotide resulting in an entirely modular system whereby any vector element can be independently exchanged. The high copy number ColE1 origin of replication has been included to enhance plasmid yield in Escherichia coli. FRT recombination sites flank the selectable marker cassette regions and allow for in planta excision by FLP recombinase. The pORE series consists of three basic types; an 'open' set for general plant transformation, a 'reporter' set for promoter analysis and an 'expression' set for constitutive expression of transgenes. The sets comprise various combinations of promoters (P (HPL), P (ENTCUP2) and P (TAPADH)), selectable markers (nptII and pat) and reporter genes (gusA and smgfp).