Normal variation in pelvic roll motion pattern during straight-line trot in hand in warmblood horses.

In horses, hip hike asymmetry, i.e. left-right difference in hip upwards movement during hind limb protraction in trot, is a crucial lameness sign. Vertical hip movements are complex, influenced by both pelvic roll and pelvic vertical motion. Veterinarians find it challenging to identify low-grade lameness, and knowledge of normal variation is a prerequisite for discerning abnormalities. This study, which included 100 clinically sound Warmblood horses, aimed to describe normal variation in pelvic roll stride patterns. Data were collected during straight-line trot in hand using optical motion capture. Stride-segmented pelvic roll data, normalised with respect to time (0-100% of the stride) and amplitude (± 0.5 of horse average stride range of motion), were modelled as a linear combination of sine and cosine curves. A sine curve with one period per stride and a cosine curve with three periods per stride explained the largest proportions of roll motion: model estimate 0.335 (p < 0.01) and 0.138 (p < 0.01), respectively. Using finite mixture models, the horses could be separated into three groups sharing common pelvic roll characteristics. In conclusion, pelvic roll motion in trot follows a similar basic pattern in most horses, yet there is significant individual variation in the relative prominence of the most characteristic features.

Differences in equine spinal kinematics between straight line and circle in trot.

Work on curved tracks, e.g. on circles, is commonplace within all forms of horse training. Horse movements in circles are naturally asymmetric, including the load distribution between inner and outer limbs. Within equestrian dressage the horse is expected to bend the back laterally to follow the circle, but this has never been studied scientifically. In the current study 12 horses were measured (optical motion capture, 100 Hz) trotting on left and right circles and on the straight without rider (soft surface). Data from markers placed along the spine indicated increased lateral bending to the inside (e.g. left bending on the left circle) of the thoracolumbar back (difference left circle vs. straight - 3.75°; right circle + 3.61°) and the neck (left - 5.23°; right + 4.80° vs. straight). Lateral bending ROM increased on the circle (+ 0.87° and + 0.62°). Individual variation in straight-circle differences was evident, but each horse was generally consistent over multiple trials. Differences in back movements between circle and straight were generally small and may or may not be visible, but accompanying changes in muscle activity and limb movements may add to the visual impression.

Kinematics of saddle and rider in high-level dressage horses performing collected walk on a treadmill.

REASONS FOR PERFORMING THE STUDY: The kinematics of the saddle and rider have not been thoroughly described at the walk. OBJECTIVE: To describe saddle and rider movements during collected walk in a group of high-level dressage horses and riders. METHODS: Seven high-level dressage horses and riders were subjected to kinematic measurements while performing collected walk on a treadmill. Movements of the saddle and rider's pelvis, upper body and head were analysed in a rigid body model. Projection angles were determined for the rider's arms and legs, and the neck and trunk of the horse. Distances between selected markers were used to describe rider position in relation to the horse and saddle. RESULTS: During the first half of each hindlimb stance the saddle rotated cranially around the transverse axis, i.e. the front part was lowered in relation to the hind part and the rider's pelvis rotated caudally, i.e. in the opposite direction. The rider's seat moved forwards while the rider's neck and feet moved backwards. During the second half of hindlimb stance these movements were reversed. CONCLUSION: The saddles and riders of high-level dressage horses follow a common movement pattern at collected walk. The movements of the saddle and rider are clearly related to the movements of the horse, both within and outside the sagittal plane. POTENTIAL RELEVANCE: The literature suggests that the rider's influence on the movement pattern of the horse is the strongest at walk. For assessment of the horse-rider interaction in dressage horses presented for unsatisfactory performance, evaluations at walk may therefore be the most rewarding. Basic knowledge about rider and saddle movements in well-performing horses is likely to be supportive to this task.

Range of motion and between-measurement variation of spinal kinematics in sound horses at trot on the straight line and on the lunge.

Clinical assessment of spinal motion in horses is part of many routine clinical exams but remains highly subjective. A prerequisite for the quantification of spinal motion is the assessment of the expected normal range of motion and variability of back kinematics. The aim of this study was to objectively quantify spinal kinematics and between -measurement, -surface and -day variation in owner-sound horses. In an observational study, twelve owner-sound horses were trotted 12 times on four different paths (hard/soft straight line, soft lunge left and right). Measurements were divided over three days, with five repetitions on day one and two, and two repetitions on day three (recheck) which occurred 28-55 days later. Optical motion capture was used to collect kinematic data. Elements of the outcome were: 1) Ranges of Motion (ROM) with confidence intervals per path and surface, 2) a variability model to calculate between-measurement variation and test the effect of time, surface and path, 3) intraclass correlation coefficients (ICC) to determine repeatability. ROM was lowest on the hard straight line. Cervical lateral bending was doubled on the left compared to the right lunge. Mean variation for the flexion-extension and lateral bending of the whole back were 0.8 and 1 degrees. Pelvic motion showed a variation of 1.0 (pitch), 0.7 (yaw) and 1.3 (roll) degrees. For these five parameters, a tendency for more variation on the hard surface and reduced variation with increased repetitions was observed. More variation was seen on the recheck (p<0.001). ICC values for pelvic rotations were between 0.76 and 0.93, for the whole back flexion-extension and lateral bending between 0.51 and 0.91. Between-horse variation was substantially higher than within-horse variation. In conclusion, ROM and variation in spinal biomechanics are horse-specific and small, necessitating individual analysis and making subjective and objective clinical assessment of spinal kinematics challenging.

Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine.

The methylated nucleoside 1-methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in all transfer RNAs (tRNAs) that read codons starting with C except in those tRNAs that read CAN codons. All of the three proline tRNA species, which read CCN codons in Salmonella typhimurium, have been sequenced and shown to contain m1G in position 37. A mutant of S. typhimurium that lacks m1G in its tRNA when grown at temperatures above 37 degrees C, has now been isolated. The mutation (trmD3) responsible for this methylation deficiency is in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. Therefore, the three proline tRNAs in the trmD3 mutant have an unmodified guanosine at position 37. Furthermore, the trmD3 mutation also causes at least one of the tRNAPro species to frequently shift frame when C's are present successively in the message. Thus, m1G appears to prevent frameshifting. The data from eubacteria apply to both eukaryotes and archaebacteria.

Basic kinematics of the saddle and rider in high-level dressage horses trotting on a treadmill.

REASONS FOR PERFORMING STUDY: A comprehensive kinematic description of rider and saddle movements is not yet present in the scientific literature. OBJECTIVE: To describe saddle and rider movements in a group of high-level dressage horses and riders. METHOD: Seven high-level dressage horses and riders were subjected to kinematic measurements while performing collected trot on a treadmill. For analysis a rigid body model for the saddle and core rider segments, projection angles of the rider's extremities and the neck and trunk of the horse, and distances between markers selected to indicate rider position were used. RESULTS: For a majority of the variables measured it was possible to describe a common pattern for the group. Rotations around the transverse axis (pitch) were generally biphasic for each diagonal. During the first half of stance the saddle rotated anti-clockwise and the rider's pelvis clockwise viewed from the right and the rider's lumbar back extended. During the later part of stance and the suspension phase reverse pitch rotations were observed. Rotations of the saddle and core rider segments around the longitudinal (roll) and vertical axes (yaw) changed direction only around time of contact of each diagonal. CONCLUSION: The saddles and riders of high-level dressage horses follow a common movement pattern at collected trot. The movements of the saddle and rider are clearly related to the movements of the horse and saddle movements also seem to be influenced by the rider. POTENTIAL RELEVANCE: Knowledge about rider and saddle movements can further our understanding of, and hence possibilities to prevent, orthopaedic injuries related to the exposure of the horse to a rider and saddle.

Back kinematics of healthy trotting horses during treadmill versus over ground locomotion.

REASONS FOR PERFORMING STUDY: Treadmill locomotion is frequently used for training of sport horses, for diagnostic purposes and for research. Identification of the possible biomechanical differences and similarities between the back movement during treadmill (T) and over ground (O) locomotion is essential for the correct interpretation of research results. OBJECTIVES: To compare the kinematics of the thoracolumbar vertebral column in treadmill and over ground locomotion in healthy horses. METHODS: Six sound Dutch Warmblood horses trotted on a T and O during 10 s at their own preferred velocity (mean +/- s.d. 3.6 +/- 0.3 m/s T and 3.6 +/- 0.1 m/s O), which was the same in both conditions. Kinematics of the vertebral column was captured by infrared cameras using reflective skin markers attached over the spinous processes of selected vertebrae and other locations. Flexion-extension and lateral bending range of motion (ROM), angular motion pattern (AMP) and intravertebral pattern symmetry (IVPS) of 5 vertebral angles (T6-T10-T13, T10-T13-T17, T13-T17-L1, T17-L1-L3 and L1-L3-15) were calculated. Neck angle, linear and temporal stride parameters and protraction-retraction angles of the limbs were also calculated. RESULTS: The vertical ROM (flexion-extension) was similar in both conditions, but the horizontal ROM (lateral bending) of the lumbar angles T17-L1-L3 and L1-L3-L5 was less during T locomotion (mean +/- s.d. difference of 1.8 +/- 0.6 and 1.7 +/- 0.9 degrees, respectively, P > 0.05). During O locomotion, the symmetry pattern of the lumbar vertebral angles was diminished from 0.9 to 0.7 (1 = 100% symmetry) indicating increased irregularity of the movement (P > 0.05). No differences were found in the basic linear and temporal stride parameters and neck angle. POTENTIAL RELEVANCE: Vertebral kinematics during treadmill locomotion is not identical to over ground locomotion, but the differences are minor. During treadmill locomotion lumbar motion is less, and caution should be therefore taken when interpreting lumbar kinematics.

The effect of different head and neck positions on the caudal back and hindlimb kinematics in the elite dressage horse at trot.

REASONS FOR PERFORMING STUDY: Dressage involves training of the horse with the head and neck placed in a position defined by the rider. The best position for dressage training is currently under debate among riders and trainers, but there are few scientific data available to confirm or disprove the different views. OBJECTIVE: To evaluate the kinematic effects of different head and neck positions (HNPs) in elite dressage horses ridden at trot. METHODS: Seven high-level dressage horses were subjected to kinetic and kinematic measurements when ridden on a treadmill with the head and neck in 5 different positions. RESULTS: Compared to free trot on loose reins the HNP desired for collected trot at dressage competitions increased T6 vertical excursion, increased sacral flexion and decreased limb retraction after lift-off. Further increasing head or head and neck flexion caused few additional changes while an extremely elevated neck position increased hindlimb flexion and lumbar back extension during stance, increased hindlimb flexion during swing and further increased trunk vertical excursion. CONCLUSIONS: The movements of the horse are significantly different when ridden on loose reins compared to the position used in collected trot. The exact degree of neck flexion is, however, not consistently correlated to the movements of the horse's limbs and trunk at collected trot. An extremely elevated neck position can produce some effects commonly associated with increased degree of collection, but the increased back extension observed with this position may place the horse at risk of injury if ridden in this position for a prolonged period. POTENTIAL RELEVANCE: Head and neck positions influence significantly the kinematics of the ridden horse. It is important for riders and trainers to be aware of these effects in dressage training.

Kinetics and kinematics of the horse comparing left and right rising trot.

REASONS FOR PERFORMING STUDY: At rising trot the rider sits alternately down on one diagonal pair of limbs and rises up on the other. The possible effects on asymmetry of locomotion induced by rising trot have rarely been studied. OBJECTIVES: To demonstrate whether, and if so to what extent, rising trot causes asymmetrical loading in the vertical ground reaction force (VGRF) and/or asymmetrical effects on the locomotion pattern, comparing left and right side. METHODS: Seven elite horses were ridden in left and right rising trot on a treadmill, while VGRF and kinematics were measured, with the horses' neck raised, the poll high and the bridge of the nose slightly in front of the vertical. RESULTS: Force loading was generally increased in the limbs of the sitting diagonal. The lumbar back was lower between mid-stances of the sitting and nonsitting stance, pelvic roll was limited and the tuber coxae heights were lower on the sitting side. Maximal hindlimb protraction was decreased. Forelimb retraction was increased and the T6 height decreased. CONCLUSION: The rider movement induces an uneven biphasic load that affects the back, pelvis and limb kinematics and VGRF. POTENTIAL RELEVANCE: The generally advocated technique of alternating limbs when riding in rising trot is supported. The VGRF changes between rising on the left or right diagonal were distinct, but minor in absolute terms and therefore unlikely to have direct impact on the occurrence of locomotor injuries. Knowledge of an increase of asymmetry in rising trot is potentially useful for riders/trainers.

Kinetics and kinematics of the passage.

REASONS FOR PERFORMING STUDY: The load acting on the limbs and the load distribution between fore- and hindlimbs while performing specific dressage exercises lack objective assessment. HYPOTHESIS: The greater a horse's level of collection, the more load is shifted to the rear and that during the passage the vertical load on the limbs increases in relation to the accentuated vertical movement of the centre of mass. METHODS: Back and limb kinematics, vertical ground reaction force and time parameters of each limb were measured in 6 Grand Prix dressage horses performing on an instrumented treadmill at the trot and the passage. Horses were ridden by their own professional rider. RESULTS: At the passage, horses moved at a slower speed (-43.2%), with a lower stride frequency (-23.6%) and, therefore, higher stride impulses (+31.0%). Relative stance duration of fore- and hindlimbs and suspension duration remained unchanged. While at the trot the diagonal limbs impacted almost simultaneously, the hindlimbs always impacted first at the passage; the time dissociation between landing and lift-off remained unchanged. Because of the prolonged stride duration, stride impulse and consequently limb impulses were higher at the passage in the fore- as well as in the hindlimbs (+24.8% and +39.9%, respectively). Within the diagonal limb pair, load was shifted from the forehand to the hindquarters (percentage stride impulse carried by the forehand -4.8%). Despite the higher impulses, peak vertical forces in the fore- and hindlimbs remained unchanged because of the prolonged absolute stance durations in fore- and hindlimbs (+28.1% and +32.2%, respectively). CONCLUSIONS: Based on the intralimb timing, the passage closely resembles the trot. Compared to other head-neck positions, the higher degree of collection resulted in a pronounced shift in impulse towards the hindquarters. Despite the higher limb impulses, peak forces acting on the limbs were similar to those observed at the trot. POTENTIAL CLINICAL RELEVANCE: An understanding of load distribution between fore- and hindlimbs in relation to different riding techniques is crucial to prevent wear-and-tear on the locomotor apparatus.

Intersegmental strategies in frontal plane in moderately-skilled riders analyzed in ridden and un-mounted situations.

The symmetry of the rider is highly relevant, and in the equestrian community it is generally thought that a symmetrical rider has a better possibility to influence the horse in an optimal way. The aim of the study was to analyse and compare frontal plane kinematics of the core body segments in ten riders while riding and while rocking a balance chair from side-to-side. It was hypothesized that the riders were asymmetrical in relation to their intersegmental strategies when comparing between left and right directions and that individual riders would display the same postural strategies when riding and when rocking the balance chair. Ten moderately-skilled riders wore a full-body marker set that was tracked by a motion capture system as they rocked a balance chair from side to side. Inertial measurement units attached to the head, trunk and pelvis were used to measure the segmental movements while riding in left and right directions. Roll rotation data for head, trunk and pelvis were averaged over available strides/cycles. Results from mixed models showed that the riders were asymmetric when comparing riding in left vs right directions, for example the trunk was rotated 19° to the right on the right circle and 14° to the left on the left circle, on average. Riders adopted the same asymmetrical posture whether they were riding in the left or right direction on straight lines, circles or leg yielding. A significant relationship was found between postural asymmetries when riding and when rocking the balance chair, one degree of pelvis or head roll asymmetry on the chair predicted 2.4 (SE 0.9) degrees of asymmetry while riding. Future studies may investigate the value of seated, off-horse postural training for improving rider symmetry and thereby equestrian performance.

Lateral movement of the saddle relative to the equine spine in rising and sitting trot on a treadmill.

Saddle slip, defined as a progressive lateral displacement of the saddle during ridden exercise, has recently been given attention in the scientific press as a potential sign of lameness. The aim of this study was to objectively quantify the normal lateral movement (oscillations) of the saddle relative to the horse in non-lame horses, and associate this movement to the movements of the horse and rider. Data from seven Warmblood dressage horses competing at Grand Prix (n = 6) or FEI Intermediate (n = 1) level, ridden by their usual riders, were used. Simultaneous kinetic, kinematic and saddle pressure measurements were conducted during sitting and rising trot on a force-measuring treadmill. The maximum lateral movement of the caudal part of the saddle relative to the horse's spine (MAX) was determined for each diagonal step. A mixed model was applied, with MAX as outcome, and T6 and S3 vertical position, rigid body rotation angles (roll, pitch, yaw) of the horse's and rider's pelvis, vertical ground reaction forces, saddle force, and rider position (rising in rising trot, sitting in rising trot or sitting in sitting trot) as explanatory variables. The least square means for MAX were 14.3 (SE 4.7) mm and 23.9 (SE 4.7) mm for rising and sitting in rising trot, and 20.3 (SE 4.7) mm for sitting trot. A 10 mm increase in maximum pelvic height at push off increased MAX by 1.4 mm (p<0.0001). One degree increase in rider pelvis roll decreased MAX 1.1 mm, and one degree increase in rider pelvis yaw increased MAX 0.7 mm (both p<0.0001). The linear relationships found between MAX and movements of both horse and rider implies that both horse and rider movement asymmetries are reflected in the lateral movements or oscillations of the saddle in non-lame horses.

A chicken beta-actin gene can complement a disruption of the Saccharomyces cerevisiae ACT1 gene.

Recently it was demonstrated that beta-actin can be produced in Saccharomyces cerevisiae by using the expression plasmid pY beta actin (R. Karlsson, Gene 68:249-258, 1988), and several site-specific mutants are now being produced in a protein engineering study. To establish a system with which recombinant actin mutants can be tested in vivo and thus enable a correlation to be made with functional effects observed in vitro, a yeast strain lacking endogenous yeast actin and expressing exclusively beta-actin was constructed. This strain is viable but has an altered morphology and a slow-growth phenotype and is temperature sensitive to the point of lethality at 37 degrees C.

Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae.

The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.

Modulation of tRNA(iMet), eIF-2, and eIF-2B expression shows that GCN4 translation is inversely coupled to the level of eIF-2.GTP.Met-tRNA(iMet) ternary complexes.

To understand how phosphorylation of eukaryotic translation initiation factor (eIF)-2 alpha in Saccharomyces cerevisiae stimulates GCN4 mRNA translation while at the same time inhibiting general translation initiation, we examined the effects of altering the gene dosage of initiator tRNA(Met), eIF-2, and the guanine nucleotide exchange factor for eIF-2, eIF-2B. Overexpression of all three subunits of eIF-2 or all five subunits of eIF-2B suppressed the effects of eIF-2 alpha hyperphosphorylation on both GCN4-specific and general translation initiation. Consistent with eIF-2 functioning in translation as part of a ternary complex composed of eIF-2, GTP, and Met-tRNA(iMet), reduced gene dosage of initiator tRNA(Met) mimicked phosphorylation of eIF-2 alpha and stimulated GCN4 translation. In addition, overexpression of a combination of eIF-2 and tRNA(iMet) suppressed the growth-inhibitory effects of eIF-2 hyperphosphorylation more effectively than an increase in the level of either component of the ternary complex alone. These results provide in vivo evidence that phosphorylation of eIF-2 alpha reduces the activities of both eIF-2 and eIF-2B and that the eIF-2.GTP. Met-tRNA(iMet) ternary complex is the principal component limiting translation in cells when eIF-2 alpha is phosphorylated on serine 51. Analysis of eIF-2 alpha phosphorylation in the eIF-2-overexpressing strain also provides in vivo evidence that phosphorylated eIF-2 acts as a competitive inhibitor of eIF-2B rather than forming an excessively stable inactive complex. Finally, our results demonstrate that the concentration of eIF-2-GTP. Met-tRNA(iMet) ternary complexes is the cardinal parameter determining the site of reinitiation on GCN4 mRNA and support the idea that reinitiation at GCN4 is inversely related to the concentration of ternary complexes in the cell.

Interactions between Ty1 retrotransposon RNA and the T and D regions of the tRNA(iMet) primer are required for initiation of reverse transcription in vivo.

Reverse transcription of the Saccharomyces cerevisiae Ty1 retrotransposon is primed by tRNA(iMet) base paired to the primer binding site (PBS) near the 5' end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNA(iMet). A structural probing study of the interactions between the Ty1 RNA template and the tRNA(iMet) primer showed that besides interactions between the PBS and the 3' end of tRNA(iMet), three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNA(iMet). To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNA(iMet) were tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNA(iMet) contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNA(iMet) and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNA(iMet).

Multiple molecular determinants for retrotransposition in a primer tRNA.

Retroviruses and long terminal repeat-containing retroelements use host-encoded tRNAs as primers for the synthesis of minus strong-stop DNA, the first intermediate in reverse transcription of the retroelement RNA. Usually, one or more specific tRNAs, including the primer, are selected and packaged within the virion. The reverse transcriptase (RT) interacts with the primer tRNA and initiates DNA synthesis. The structural and sequence features of primer tRNAs important for these specific interactions are poorly understood. We have developed a genetic assay in which mutants of tRNA(iMet), the primer for the Ty1 retrotransposon of Saccharomyces cerevisiae, can be tested for the ability to serve as primers in the reverse transcription process. This system allows any tRNA mutant to be tested, regardless of its ability to function in the initiation of protein synthesis. We find that mutations in the T psi C loop and the acceptor stem regions of the tRNA(iMet) affect transposition most severely. Conversely, mutations in the anticodon region have only minimal effects on transposition. Further study of the acceptor stem and other mutants demonstrates that complementarity to the element primer binding site is a necessary but not sufficient requirement for effective tRNA priming. Finally, we have used interspecies hybrid initiator tRNA molecules to implicate nucleotides in the D arm as additional recognition determinants. Ty3 and Ty1, two very distantly related retrotransposons, require similar molecular determinants in this primer tRNA for transposition.

TRM6/61 connects PKCα with translational control through tRNAi(Met) stabilization: impact on tumorigenesis.

Accumulating evidence suggests that changes of the protein synthesis machinery alter translation of specific mRNAs and participate in malignant transformation. Here we show that protein kinase C α (PKCα) interacts with TRM61, the catalytic subunit of the TRM6/61 tRNA methyltransferase. The TRM6/61 complex is known to methylate the adenosine 58 of the initiator methionine tRNA (tRNAi(Met)), a nuclear post-transcriptional modification associated with the stabilization of this crucial component of the translation-initiation process. Depletion of TRM6/61 reduced proliferation and increased death of C6 glioma cells, effects that can be partially rescued by overexpression of tRNAi(Met). In contrast, elevated TRM6/61 expression regulated the translation of a subset of mRNAs encoding proteins involved in the tumorigenic process and increased the ability of C6 cells to form colonies in soft agar or spheres when grown in suspension. In TRM6/61/tRNAi(Met)-overexpressing cells, PKCα overexpression decreased tRNAi(Met) expression and both colony- and sphere-forming potentials. A concomitant increase in TRM6/TRM61 mRNA and tRNAi(Met) expression with decreased expression of PKCα mRNA was detected in highly aggressive glioblastoma multiforme as compared with Grade II/III glioblastomas, highlighting the clinical relevance of our findings. Altogether, we suggest that PKCα tightly controls TRM6/61 activity to prevent translation deregulation that would favor neoplastic development.

The yeast initiator tRNAMet can act as an elongator tRNA(Met) in vivo.

Saccharomyces cerevisiae uses two different methionine accepting tRNAs during protein synthesis. One, tRNA(iMet), is used exclusively during the initiation of translation whereas the other, tRNA(mMet), is used during the elongation of translation. To study the unique features of each methionine tRNA species, we constructed yeast strains with null alleles of the five elongator methionine tRNA (EMT) genes and strains with null alleles of the four initiator methionine tRNA (IMT) genes, respectively. Consequently, growth of these strains was dependent either on a tRNA(mMet) or a tRNA(iMet), respectively, encoded from a plasmid-derived gene. For both null mutants, the plasmid carrying the wild-type gene can be selected against and exchanged for another plasmid derived EMT or IMT gene (wild-type or mutant). A high gene dosage of the wild-type IMT gene could restore growth to the elongator-depleted strain. However, wild-type EMT genes in a high gene dosage never restored growth of the initiator depleted strain. Thus, the elongator tRNA(Met) is much more restricted to participate in the initiation of translation than the initiator tRNA(Met) is restricted to participate in the elongation process. Using the two null mutants, we have identified tRNA(mMet) mutants, which show reduced elongator activity, and tRNA(iMet) mutants, with improved elongator activity in the elongator depleted strain. Also, tRNA(mMet) mutants that function as an initiator tRNA in the initiator depleted strain were identified. From this mutant analysis, we showed that the conserved U/rT at position 54 of the elongator tRNA(Met) is an important determinant for an elongator tRNA. The most important determinant for an initiator was shown to be the acceptor stem and especially the conserved A1.U72 base-pair. Mutant tRNAs, with reduced activity in either process, were investigated for enhanced activity during overproduction of the alpha and beta-subunits of the eukaryotic initiation factor 2 (eIF-2) or the eukaryotic elongation factor 1 alpha (eEF-1 alpha). The data suggest that the U/rT of the elongator at position 54 is important for eEF-1 alpha recognition and that the acceptor stem of the initiator is important for eIF-2 recognition.

Non-autogenous control of ribosomal protein synthesis from the trmD operon in Escherichia coli.

The trmD operon of Escherichia coli encodes the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and a 21,000 Mr protein of unknown function. Here we demonstrate that, in contrast to the expression of other ribosomal protein operons, the amount of trmD operon mRNA and the rate of synthesis of the proteins encoded by the operon respond to increased gene dosage. The steady-state level of the mRNA was about 18 times higher, and the relative rate of synthesis of the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and the 21,000 Mr protein was 15, 9, 25 and 23 times higher, respectively, in plasmid-containing cells than in plasmid-free cells. Overproduced tRNA(m1G37)methyltransferase and 21,000 Mr protein were as stable as E. coli total protein, whereas the two ribosomal proteins were degraded to a large extent. The steady-state amount of S16 and L19 in the plasmid-containing cells exceeded that in plasmid-free cells by threefold and twofold, respectively. No significant effect on the synthesis of the trmD operon proteins from the chromosomally located genes was observed when parts of the operon were expressed on different plasmids. Taken together, these results suggest that the expression of the trmD operon is not subject to transcriptional or translational feedback regulation, and demonstrate that not all ribosomal protein operons are regulated in the same manner. We propose that ribosomal protein operons that do not encode proteins that bind directly to rRNA are not under autogenous control. Metabolic regulation at the transcriptional level and protein degradation are plausible mechanisms for the control of expression of such operons.