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Biomedical subjects

Markus Hossbach

Publications and source records attributed to Markus Hossbach.

7 recordsLinked to original sources
PubMed2006

Identification, cloning, and functional analysis of the human U6 snRNA-specific terminal uridylyl transferase.

Mammalian cells contain a highly specific terminal uridylyl transferase (TUTase) that exclusively accepts U6 snRNA as substrate. This enzyme, termed U6-TUTase, was purified from HeLa cell extracts and analyzed by microsequencing. All sequenced peptides matched a unique human cDNA coding for a previously unknown protein. Domain structure analysis revealed that the U6-TUTase also belongs to the well-characterized poly(A) polymerase protein superfamily. However, by amino acid sequence as well as RNA-binding motifs, human U6-TUTase is highly divergent from both the poly(A) polymerases and from the TUTases identified within the editing complexes of trypanosomes. After cloning, the recombinant U6-TUTase was expressed in HeLa cells. Analysis of its catalytical activity confirmed the identity of the cloned protein as U6-TUTase, exhibiting the same exclusive substrate specificity for U6 snRNA as the endogenous enzyme. That unique selectivity even excluded as substrate U6atac RNA, the functional homolog of the minor spliceosome. Finally, RNAi knockdown experiments revealed that U6-TUTase is essential for cell proliferation. Surprisingly, large amounts of the recombinant enzyme were found to accumulate within nucleoli.

Amino Acid Motifs↗
PubMed2006

Gene silencing with siRNA duplexes composed of target-mRNA-complementary and partially palindromic or partially complementary single-stranded siRNAs.

Synthetic small interfering RNA (siRNA) duplexes are widely used to transiently and sequence-specifically disrupt gene expression in mammalian cultured cells. The efficiency and specificity of mRNA cleavage is partly affected by the presence of the nontargeting "passenger" or "sense" siRNA strand, which is required for presentation of the target-complementary or guide siRNA strand to the double-strand-specific RNA silencing protein machinery. We show that siRNA duplexes can be designed that are solely composed of two fully target-complementary guide strands that are sufficiently complementary to each other to form stable duplexes with characteristic 3' overhanging ends. The general feasibility of this approach is documented by transient knockdown of lamin A/C and emerin in HeLa cells. The silencing efficiencies of guide-only siRNA duplexes are comparable to prototypical fully paired passenger/guide duplex siRNAs, even though guide-only siRNA duplexes may contain a significant number of nonWatson-Crick and G/U wobble base pairs. Such siRNA duplexes may offer advantages regarding production costs and specificity of gene silencing.

Base Sequence↗
PubMed2004

Assembly and maturation of the U3 snoRNP in the nucleoplasm in a large dynamic multiprotein complex.

The assembly and maturation of box C/D snoRNPs, factors essential for ribosome biogenesis, occur in the nucleoplasm. To investigate this process, we have analyzed non-snoRNP factors associated with the nucleoplasmic human U3 snoRNA. We show that both the precursor and mature length nucleoplasmic U3 snoRNAs are present in larger multiprotein complexes that contain the core box C/D proteins as well as many non-snoRNP factors linked to snoRNP assembly (TIP48, TIP49, Nopp140), RNA processing (TGS1, La, LSm4, hRrp46), and subcellular localization (CRM1, PHAX). Using RNAi, we show that most of these factors are essential for box C/D snoRNA accumulation. Furthermore, we demonstrate that the core proteins undergo a restructuring event that stabilizes their binding to the snoRNA. Importantly, restructuring, which may be mediated by the putative remodeling factor TIP49, appears to be linked to nucleolar localization. We believe that the assembly complex coordinates snoRNA processing, snoRNP assembly, restructuring, and localization.

Base Sequence↗
PubMed2004

RNAi knockdown of hPrp31 leads to an accumulation of U4/U6 di-snRNPs in Cajal bodies.

Cajal bodies (CBs) are subnuclear organelles of animal and plant cells. A role of CBs in the assembly and maturation of small nuclear ribonucleoproteins (snRNP) has been proposed but is poorly understood. Here we have addressed the question where U4/U6.U5 tri-snRNP assembly occurs in the nucleus. The U4/U6.U5 tri-snRNP is a central unit of the spliceosome and must be re-formed from its components after each round of splicing. By combining RNAi and biochemical methods, we demonstrate that, after knockdown of the U4/U6-specific hPrp31 (61 K) or the U5-specific hPrp6 (102 K) protein in HeLa cells, tri-snRNP formation is inhibited and stable U5 mono-snRNPs and U4/U6 di-snRNPs containing U4/U6 proteins and the U4/U6 recycling factor p110 accumulate. Thus, hPrp31 and hPrp6 form an essential connection between the U4/U6 and U5 snRNPs in vivo. Using fluorescence microscopy, we show that, in the absence of either hPrp31 or hPrp6, U4/U6 di-snRNPs as well as p110 accumulate in Cajal bodies. In contrast, U5 snRNPs largely remain in nucleoplasmic speckles. Our data support the idea that CBs may play a role in tri-snRNP recycling.

Carrier Proteins↗
PubMed2004

siRNA Selection Server: an automated siRNA oligonucleotide prediction server.

The Whitehead siRNA (short interfering RNA) Selection Web Server (http://jura.wi.mit.edu/bioc/siRNA) automates the design of short oligonucleotides that can specifically 'knock down' expression of target genes. These short sequences are about 21 nt in length, and when synthesized as double stranded RNA and introduced into cell culture, can reduce or eliminate the function of the target gene. Depending on the length of a gene, there are potentially numerous combinations of possible 21mers. Some experimental evidence has already shown that not all 21mers in a gene have the same effectiveness at silencing gene function. Our tool incorporates published design rules and presents the scientist with information about uniqueness of the 21mers within the genome, thermodynamic stability of the double stranded RNA duplex, GC content, presence of SNPs and other features that may contribute to the effectiveness of a siRNA.

Algorithms↗
PubMed2004

The human 18S U11/U12 snRNP contains a set of novel proteins not found in the U2-dependent spliceosome.

U11 and U12 snRNPs bind U12-type pre-mRNAs as a preformed di-snRNP complex, simultaneously recognizing the 5' splice site and branchpoint sequence. Thus, within the U12-type prespliceosome, U11/U12 components form a molecular bridge connecting both ends of the intron. We have affinity purified human 18S U11/U12 and 12S U11 snRNPs, and identified their protein components by using mass spectrometry. U11/U12 snRNPs lack all known U1 snRNP proteins but contain seven novel proteins (i.e., 65K, 59K, 48K, 35K, 31K, 25K, 20K) not found in the major spliceosome, four of which (59K, 48K, 35K, and 25K) are U11-associated. Thus, protein-protein and protein-RNA interactions contributing to 5' splice site recognition and/or intron bridging appear to differ significantly in the minor versus major prespliceosome. The majority of U11/U12 proteins are highly conserved in organisms known to contain U12-type introns. However, homologs of those associated with U11 were not detected in Drosophila melanogaster, consistent with the presence of a divergent U11 snRNP in flies. RNAi experiments revealed that several U11/U12 proteins are essential for cell viability, suggesting they play key roles in U12-type splicing. The presence of unique U11/U12 snRNP proteins in the U12-type spliceosome provides insight into potential evolutionary relationships between the major and minor spliceosome.

Amino Acid Sequence↗