Giuseppe Biamonti

A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates

A method is described for the absolute quantification by polymerase chain reaction (PCR) of nucleic acids present in low abundance. The method entails the addition to the sample of competitor DNA molecules that share the same sequence as the amplified target (including primer recognition sites), except for a 20-bp insertion in the middle, which allows easy resolution by gel electrophoresis (competitive PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial rate of targets, rendering the reaction independent of the number of amplification cycles. An easy and reliable method for the construction and quantification of competitive templates obtained as recombinant PCR products was developed. The technique was used for the absolute quantification of human genomic DNA with primers from a single copy, subtelomeric region of chromosome 19.

Cellular stress and RNA splicing

In response to physical and chemical stresses that affect protein folding and, thus, the execution of normal metabolic processes, cells activate gene-expression strategies aimed at increasing their chance of survival. One target of several stressing agents is pre-mRNA splicing, which is inhibited upon heat shock. Recently, the molecular basis of this splicing inhibition has begun to emerge. Interestingly, different mechanisms seem to be in place to block constitutive pre-mRNA splicing and to affect alternative splicing regulation. This could be important to modulate gene expression during recovery from stress. Thus, pre-mRNA splicing emerges as a central mechanism to integrate cellular and metabolic stresses into gene-expression profiles.

Sam68 regulates EMT through alternative splicing–activated nonsense-mediated mRNA decay of the SF2/ASF proto-oncogene

Expression levels of SF2/ASF are controlled by Sam68 mediated activation of splicing-induced mRNA decay.

Alternative splicing and tumor progression.

Alternative splicing is a key molecular mechanism for increasing the functional diversity of the eukaryotic proteomes. A large body of experimental data implicates aberrant splicing in various human diseases, including cancer. Both mutations in cis-acting splicing elements and alterations in the expression and/or activity of splicing regulatory factors drastically affect the splicing profile of many cancer-associated genes. In addition, the splicing profile of several cancer-associated genes is altered in particular types of cancer arguing for a direct role of specific splicing isoforms in tumor progression. Deciphering the mechanisms underlying aberrant splicing in cancer may prove crucial to understand how splicing machinery is controlled and integrated with other cellular processes, in particular transcription and signaling pathways. Moreover, the characterization of splicing deregulation in cancer will lead to a better comprehension of malignant transformation. Cancer-associated alternative splicing variants may be new tools for the diagnosis and classification of cancers and could be the targets for innovative therapeutical interventions based on highly selective splicing correction approaches.

Transcription of Satellite III non-coding RNAs is a general stress response in human cells

In heat-shocked human cells, heat shock factor 1 activates transcription of tandem arrays of repetitive Satellite III (SatIII) DNA in pericentromeric heterochromatin. Satellite III RNAs remain associated with sites of transcription in nuclear stress bodies (nSBs). Here we use real-time RT-PCR to study the expression of these genomic regions. Transcription is highly asymmetrical and most of the transcripts contain the G-rich strand of the repeat. A low level of G-rich RNAs is detectable in unstressed cells and a 104-fold induction occurs after heat shock. G-rich RNAs are induced by a wide range of stress treatments including heavy metals, UV-C, oxidative and hyper-osmotic stress. Differences exist among stressing agents both for the kinetics and the extent of induction (>100- to 80.000-fold). In all cases, G-rich transcripts are associated with nSBs. On the contrary, C-rich transcripts are almost undetectable in unstressed cells and modestly increase after stress. Production of SatIII RNAs after hyper-osmotic stress depends on the Tonicity Element Binding Protein indicating that activation of the arrays is triggered by different transcription factors. This is the first example of a non-coding RNA whose transcription is controlled by different transcription factors under different growth conditions.

Modular Structure of the Human Lamin B2 Replicator

ABSTRACT The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation.

Nuclear Stress Bodies

Nuclear stress bodies (nSBs) are unique subnuclear organelles which form in response to heat shock. They are initiated through a direct interaction between heat shock transcription factor 1 (HSF1) and pericentric tandem repeats of satellite III sequences and correspond to active transcription sites for noncoding satellite III transcripts. Given their unusual features, nSBs are distinct from other known transcription sites. In stressed cells, they are thought to participate in rapid, transient, and global reprogramming of gene expression through different types of mechanisms including chromatin remodeling and trapping of transcription and splicing factors. The analysis of these atypical and intriguing structures uncovers new facets of the relationship between nuclear organization and nuclear function.

Early mitotic degradation of the homeoprotein HOXC10 is potentially linked to cell cycle progression

Hox proteins are transcription factors involved in controlling axial patterning, leukaemias and hereditary malformations. Here, we show that HOXC10 oscillates in abundance during the cell cycle, being targeted for degradation early in mitosis by the ubiquitin‐dependent proteasome pathway. Among abdominal‐B subfamily members, the mitotic proteolysis of HOXC10 appears unique, since the levels of the paralogous HOXD10 and the related homeoprotein HOXC13 are constant throughout the cell cycle. When two destruction box motifs (D‐box) are mutated, HOXC10 is stabilized and cells accumulate in metaphase. HOXC10 appears to be a new prometaphase target of the anaphase‐promoting complex (APC), since its degradation coincides with cyclin A destruction and is suppressed by expression of a dominant‐negative form of UbcH10, an APC‐associated ubiquitin‐conjugating enzyme. Moreover, HOXC10 co‐immunoprecipitates the APC subunit CDC27, and its in vitro degradation is reduced in APC‐depleted extracts or by competition with the APC substrate cyclin A. These data imply that HOXC10 is a homeoprotein with the potential to influence mitotic progression, and might provide a link between developmental regulation and cell cycle control.

Mammalian single-stranded DNA binding protein UP I is derived from the hnRNP core protein A1.

Antibodies induced against mammalian single‐stranded DNA binding protein (ssDBP) UP I were shown to be cross‐reactive with most of the basic hnRNP core proteins, the main constituents of 40S hnRNP particles. This suggested a structural relationship between both groups of proteins. Using the anti‐ssDBP antibodies, a cDNA clone (pRP10) was isolated from a human liver cDNA library in plasmid expression vector pEX1. By DNA sequencing this clone was shown to encode in its 949 bp insert the last 72 carboxy terminal amino acids of the ssDBP UP I. Thereafter, an open reading frame continued for another 124 amino acids followed by a UAA (ochre) stop codon. Direct amino acid sequencing of a V8 protease peptide from hnRNP core protein A1 showed that this peptide contained at its amino terminus the last 11 amino acids of UP I followed by 19 amino acids which are encoded by the open reading frame of cDNA clone pRP10 immediately following the UP I sequence. This proves that ssDBP UP I arises by proteolysis from hnRNP core protein A1. This finding must lead to a re‐evaluation of the possible physiological role of UP I and related ssDBPs. The formerly assumed function in DNA replication, although not completely ruled out, should be reconsidered in the light of a possible alternative or complementary function in hnRNA processing where UP I could either be a simple degradation product of core protein A1 (as a consequence of controlling the levels of active A1) or may continue to function as an RNA binding protein which has lost the ability to interact with the other core proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear stress bodies: a heterochromatin affair?

It is still largely unknown how the various nuclear subcompartments are formed, why they form in particular locations and how they are linked to nuclear function. Nuclear stress bodies provide a new opportunity to address these questions and to test models of self-organization of nuclear structures. The assembly of these bodies requires the synthesis of non-coding RNAs with a probable role in the post-transcriptional regulation of gene expression and in higher-order chromatin organization.